Fibronectin-functionalization of 3D collagen networks supports immune tolerance and inflammation suppression in human monocyte-derived macrophages

Abstract

The extracellular matrix (ECM) is dynamically reorganized during wound healing. Concomitantly, recruited monocytes differentiate into macrophages. However, the role of the wound's ECM during this transition remain to be fully understood. Fibronectin is a multifunctional glycoprotein present in early wound ECM with a potential immunomodulatory role during monocyte-to-macrophage differentiation. Hence, to investigate the impact of fibronectin during this differentiation step, 3D fibrillar collagen type I networks with or without fibronectin-functionalization were engineered with defined topology (fibril and pore diameter: 0.8 μm; 7 μm) and amount of adsorbed fibronectin (0.15 μg per μg collagen). Primary, human monocytes were then differentiated into macrophages inside these networks. The immunological imprinting of the resulting macrophages was monitored by means of the expression of FABP4, CLEC4E, SLC2A6, and SOD2 which discriminate naïve and tolerized macrophages, as well pro-inflammatory (M1) and anti-inflammatory (M2) macrophage polarization. The analyses indicate that fibronectin-functionalization of collagen I networks induces macrophage tolerance rather than M1 or M2 macrophage phenotypes. This finding was confirmed by release profiles of pro- and anti-inflammatory cytokines such as IL6, IL8, CXCL10, and IL10. Nevertheless, upon LPS challenge, immune suppression by fibronectin was overridden since these macrophages could then deploy an efficient immune response. Our results therefore provide new perspectives in biomaterial science of wound healing scaffolds and the design of instructive materials for human monocyte-derived cells.

Introduction

Wound healing is a highly organized process proceeding through a sequence of overlapping phases including recruitment and proliferation of different immune cells, deposition of extracellular matrix (ECM), and remodeling of newly assembled tissue [1]. At the same time, ECM cues tightly control cell migration, proliferation, and differentiation [[2], [3], [4]]. Thus, dynamic changes in ECM composition, elasticity and microstructure are presumed driving forces of healing progress and success [[5], [6], [7]]. Following wounding, circulating blood monocytes are actively recruited to wound sites where they differentiate into macrophages (MPh) [8]. The underlying processes during the transition from monocyte to functionally polarized MPh and the role of the wound's ECM in these processes remain to be fully understood. MPh in the wound milieu may exhibit different phenotypes ranging from classically activated MPh (M1) and regulatory MPh (M2) to presumed wound-healing MPh states. These different MPh polarization states are coherent with defined functions, including specific cytokine secretion, matrix resolution, and moderation of tissue regeneration and scar formation [9,10]. Concomitantly, immunological imprinting of tolerance or trained immunity as observed after microbe-associated molecular patterns as is the case during infection or vaccination, also determine MPh polarization [[11], [12], [13]]. Importantly, exposure of fresh monocytes to environmental cues prior to their differentiation into MPh can polarize the resulting MPh [11,12]. Hence it seems reasonable to hypothesize that the wound ECM may polarize monocyte-derived MPh towards a wound-healing phenotype. Furthermore, a promising approach in regenerative medicine is to modulate the macrophage function and thus the tissue repair progress by biomaterial properties [14]. For this reason, detailed knowledge about influences of the ECM on the MPh polarization may improve in vitro models of wound healing processes in medical applications as well as provide further insights into details of in vivo wound healing mechanisms. In addition, the new insights can be applied to engineer new instructive materials for improved wound healing.

The dynamic transition of the ECM composition throughout the time course of healing was recently summarized [15,16]. During the first hours of injury, a hyaluronan- and plasma fibronectin-rich fibrin clot is formed that is transformed into a provisional matrix composed of tissue fibronectin and collagen types III and I. The provisional matrix is further remodeled into scar tissue preferentially composed of collagen type I and glycosaminoglycans [6,17]. While collagen type I is present in the provisional matrix and the scar tissue; fibronectin is present in the initial fibrin clot and the provisional matrix. As fibronectin is present during different stages of wound healing, its potential role in immunomodulation is of high interest.

Immunosuppressive functions of fibronectin have been reported in ovarian cancer [18] and fibronectin was suggested as a source of immunosuppressive peptides during injury [19]. Furthermore, immunosuppressive/tolerizing functions of matrix molecules could convey a tissue integrity signal to immune cells and effectively communicate an “all-clear” signal to support wound healing [20]. Fibronectin is an important ECM glycoprotein that forms fibrillar networks and promotes cell adhesion by providing integrin binding sites for specific integrins such as α₅β₁ [21]. Adhesion to fibronectin furthermore activates downstream pathways to modulate cell migration, proliferation, and differentiation [21,22]. Fibronectin consists of three modular repeating units organized into larger domains with distinct functions [23]. Fibronectin also has binding sites for collagen, heparin, and fibrin, underlying its function as an ECM cross-linking molecule. Additionally, fibronectin is known to bind, present, and regulate several cytokines, including TGF-β1, VEGF, HGF, and BMP2 [2,24] and fibronectin based hydrogels successfully demonstrated functional effects in angiogenesis and bone regeneration [25]. Fibronectin's importance during wound healing has been reviewed [17,26]. Two forms of fibronectin exist: plasma fibronectin and tissue fibronectin. Soluble plasma fibronectin is produced by hepatocytes and has importance during early wound healing throughout fibrin clot formation. Tissue fibronectin is synthesized by fibroblasts, endothelial cells, and keratinocytes and occurs at later wound healing stages, when granulation tissue is formed. However, Moretti et al., 2007 detected that the major fraction of fibronectin in tissues is derived from plasma fibronectin [27]. Furthermore, in early wound healing, fibronectin can opsonize ECM debris, enabling debris-phagocytosis by MPh and the presence of fibronectin at wound edges is important for cell migration into the wound bed [17].

As pointed out above, a specific cell type of interest in wound healing are MPh and their polarization states and functions [9]. For in vitro cell culture studies, MPh polarization protocols have been applied for decades, exploiting several methodologies. For instance, after 6 days of differentiation in tissue culture dishes in the presence of macrophage colony-stimulating factor (M-CSF), circulating monocyte-derived MPh are called ‘resting’ or ‘naïve’ (M0) and can be polarized towards a ‘pro-inflammatory’ (M1) state by acute exposure to lipopolysaccharide (LPS) [28]. Alternatively, exposure of differentiating monocytes to IL4 or IL13 and rosiglitazone results in the so-called M2 ‘tissue repair’ or ‘anti-inflammatory’ state that resembles dendritic cell functionality [28]. Furthermore, in a 6-day memory setup, monocyte exposure to LPS during the first day of their differentiation towards MPh results in the generation of so-called ‘tolerized’ MPh (LPS-MPh) that lack certain pro-inflammatory properties upon re-exposure to pathogen-associated molecular patterns such as LPS- and Pam3Cis-stimulated IL6 and TNFα secretion, resembling a ‘paralyzed’ MPh state found in vivo during persistent inflammation-induced clinical sepsis [11,12]. Such properties of tolerized MPh could prove quite beneficial for the decay of exaggerated inflammatory conditions threatening to destroy host tissue, as well as for supporting of wound healing. Another clinically important MPh polarization state is displayed by so called ‘trained’ MPh (BG-MPh), which secrete more IL6 and TNFα upon Toll-like receptor stimulation [13]. MPh training can be induced to varying extents by monocyte exposure to beta-glucans, BCG vaccine, cholesterol, and some intermediate metabolites during the first 24 h of monocyte differentiation [29,30]. In the present work, we introduce the operationally-coined ‘collagen-polarized’ MPh (Coll-MPh) and the ‘collagen-fibronectin-polarized’ MPh (CollFN-MPh) whereby monocyte-to-MPh differentiation is performed inside engineered 3D collagen and collagen-fibronectin networks in the presence of exogenously added M-CSF.

To investigate the role of the ECM composition at wound sites on the MPh function, physiologically relevant model systems are needed. Several in vivo studies based on animal models revealed whole-system responses to wounding [[31], [32], [33]]. However, the high complexity of in vivo wounds hinders mechanistic dissection of causes and effects. Experiments using 2D materials in vitro have reduced complexity, but typically lack relevant mechanical and topological features, in particular a 3D organized cell microenvironment, and fail to mimic in vivo wound sites. To overcome these limitations, 3D in vitro cell cultures are increasingly applied because they permit control of the cellular and extracellular components of the system. Collagen type I, as the most abundant structural matrix protein in tissues, is a useful tool in engineering 3D networks for in vitro cell culture and it is already in use as a biomaterial for wound healing [34]. Fibrillar scaffolds made from collagen type I can be adjusted in topology, mechanic, and composition and allow control over relevant parameters of a specific wound phase [[35], [36], [37]]. 3D fibrillar collagen I networks are therefore considered as relevant scaffolds for tissue engineering and regenerative medicine that support cell interactions and integration of environmental molecular cues. Earlier studies indicate that collagen-based scaffolds induce only minimal immune responses when implanted in the body [[38], [39], [40]]. Furthermore, and important for the work presented below, collagen type I networks can be functionalized by fibronectin due to fibronectin's binding sites specific for collagen type I. It was already shown that functionalization of collagen networks with fibronectin is stable and that variable amounts of fibronectin can be introduced without modifying the mechanical properties of the scaffold [36].

On the basis of these considerations, we set out to study the immunomodulatory effect of fibronectin during human monocyte-to-MPh differentiation. 3D fibrillar networks based on collagen type I with a defined topology and optional fibronectin functionalization were prepared. Fibronectin-dependent immunological imprinting on human monocyte-derived MPh was investigated at the level of gene expression and cytokine release by Coll-MPh and CollFN-MPh, revealing a tolerant phenotype that could be overridden by LPS challenge.