Regulation of lung injury and repair by Toll-like receptors and hyaluronan

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.     

Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2

Inflammation under sterile conditions is not well understood despite its importance in trauma and autoimmune disease. To investigate this process we established mouse models of sterile injury and explored the role of hyaluronan in mediating inflammation following injury. The response of cultured monocytes to hyaluronan was different than the response to lipopolysaccharide (LPS) despite both being dependent on Toll-like receptor 4 (TLR4). Cultured cells exposed to hyaluronan showed a pattern of gene induction that mimics the response seen in mouse skin after sterile injury with an increase in molecules such as transforming growth factor-beta2 and matrix metalloproteinase-13. These factors were not induced by LPS despite the mutual dependence of both hyaluronan and LPS on TLR4. Explanation for the unique response to hyaluronan was provided by observations that a lack of TLR4 or CD44 in mice diminished the response to sterile injury, and together with MD-2, was required for responsiveness to hyaluronan in vitro. Thus, a unique complex of TLR4, MD-2, and CD44 recognizes hyaluronan. Immunoprecipitation experiments confirmed the physical association of TLR4 and CD44. Taken together, our results define a previously unknown mechanism for initiation of sterile inflammation that involves recognition of released hyaluronan fragments as an endogenous signal of tissue injury.     

Hyaluronan receptors involved in cytokine induction in monocytes

During inflammation, lower molecular weight fragments of hyaluronanaccumulate, and this is known to be inflammatory and immune-stimulatory.In diseases such as inflammatory bowel disease, inflammatorycells bind to hyaluronan; however, the cellular response andmolecular mechanism of hyaluronan–hyaluronan receptorinteractions in mononuclear cells are not well understood. Theexpression of hyaluronan receptors in peripheral blood mononuclearcells (PBMC) was examined. PBMC were stimulated with lower andhigher molecular weight hyaluronan (molecular weight 100–150kDa and 2700 kDa) and the induction of proinflammatory cytokines(interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1))was compared by enzyme-linked immunoabsorbant assay (ELISA).Cells were coincubated with various signaling pathway inhibitors.In addition, neutralizing antibodies against CD44 and TLR4 wereadded and the effects on PBMC were investigated. Finally, mononuclearcells from CD44-null and toll-like receptor 4 (TLR4) mutantmice were both stimulated with lower molecular weight hyaluronan.Among the hyaluronan receptors, TLR4 and CD44 were markedlyexpressed on PBMC. Hyaluronan-stimulated PBMC enhanced the attachmentto the extracellular matrix. Lower molecular weight hyaluronaninduced IL-6 and MCP-1 production in PBMC, but high-molecular-weighthyaluronan did not induce IL-6 and MCP-1 production. An anti-CD44antibody attenuated the induction of both IL-6 and MCP-1 inlower molecular weight hyaluronan-stimulated PBMC. In both TLR4mutant and CD44-null mice, the induction of IL-6 by lower molecularweight hyaluronan stimulation was decreased. SB203580 completelyabolished IL-6 production in both TLR4 mutant and CD44-nullmononuclear cells, while PD98059 abolished IL-6 production inCD44-null mononuclear cells. Hyaluronan receptors, CD44 andTLR4, play distinct roles in cytokine induction in hyaluronan-stimulatedmononuclear cells.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

Hyaluronan fragments: an information-rich system

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-D-glucuronic acid-beta1,3-N-acetyl-D-glucosamine-beta1,4-]n. Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell-cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2 x 10(4) kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous "danger signals", while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.     

Effects of soluble factors and extracellular matrix components on vascular cell behavior in vitro and in vivo: models of de-endothelialization and repair.

Vessel walls are comprised of several different cell populations residing in and on complex extracellular matrices. Each of the vascular cell types has diverse and sometimes unique functions and morphologies, and each has roles in repair processes following injury. Large vessel endothelial cells are known to respond to denudation injury by sheet migration and proliferation. This is in contrast to the migration through soft tissues with tube formation and subsequent lumen formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells of larger vessels respond to injury by migration from the arterial media into the intima, proliferation, and matrix biosynthesis, ultimately causing intimal thickening. Both these cell types exhibit "dysfunctional" phenotypes during their responses to injury. Microvascular cell responses to injury, while extremely variable, are less well documented. Specifically, responses to injury by microvascular endothelial vascular cells appear to be modulated, in part, by the composition and organization of the surrounding matrix as well as by the various soluble factors and cytokines found at sites of injury, suggesting that the extracellular matrix and soluble factors modulate each other's effects on local vascular cell populations following injury.     

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization

The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages, and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFkappaB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFkappaB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs) localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.     

Hyaluronic acid fragments evoke Kupffer cells via TLR4 signaling pathway

Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we investigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, dependent on p38 MAPK activation. These anthors contributed equally to this work Supported by the National Natural Science Foundation of China (Grant No. 30500487 and 30700792)     

The role of Toll-like receptors in non-infectious lung injury

The role of Toll-like receptors （TLRs） in pathogen recognition has been expeditiously advanced in recent years. However, investigations into the function of TLRs in non-infectious tissue injury have just begun. Previously, we and others have demonstrated that fragmented hyaluronan （HA） accumulates during tissue injury. CD44 is required to clear HA during tissue injury, and impaired clearance of HA results in unremitting inflammation. Additionally, fragmented HA stimulates the expression of inflammatory genes by inflammatory cells at the injury site. Recently, we identified that HA fragments require both TLR2 and TLR4 to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. In a non-infectious lung injury model, mice deficient in both TLR2 and TLR4 show an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis, and decreased survival. Lung epithelial cell overexpression of high molecular mass HA protected mice against acute lung injury and apoptosis, in part through TLR-dependent basal activation of NF-κB. The exaggerated injury in TLR2 and TLR4 deficient mice appears to be due to impaired HA-TLR interactions on epithelial cells. These studies identify that host matrix component HA and TLR interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute lung injury.     

Hyaluronan signaling during ozone-induced lung injury requires TLR4, MyD88, and TIRAP

Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone-induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4-/-, MyD88-/-, and TIRAP-/- mice were characterized for AHR after exposure to either ozone (1 ppm × 3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4-/-, MyD88-/-, or TIRAP-/- mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4-/-, MyD88-/-, or TIRAP-/- mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway.     

Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4

Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Here, we report that syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair.     

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.     

Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.     

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.     

Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.     

Hyaluronan and its catabolic products in tissue injury and repair.

Hyaluronan is an unbiquitous glycosaminoglycan present in most tissues. Under homeostatic conditions hyaluronan exists as a high molecular mass polymer that has important roles in tissue structural integrity. Under conditions of stress such as following tissue injury, hyaluronan becomes depolymerized and lower molecular mass polymers are generated. The biological properties of these hyaluronan fragments appear to be distinct from the larger precursor molecules. This review examines the biological role of hyaluronan fragments in tissue injury and repair.     

Tissue repair and the dynamics of the extracellular matrix

Repair of tissue after injury depends on the synthesis of a fibrous extracellular matrix to replace lost or damaged tissue. Newly deposited extracellular matrix is then re-modeled over time to emulate normal tissue. The extracellular matrix directs repair by regulating the behavior of the wide variety of cell types that are mobilized to the damaged area in order to rebuild the tissue. Acute inflammation, re-epithelialization, and contraction all depend on cell-extracellular matrix interactions and contribute to minimize infection and promote rapid wound closure. Matricellular proteins are up-regulated during wound healing where they modulate interactions between cells and the extracellular matrix to exert control over events that are essential for efficient tissue repair. Here, we discuss how the extracellular matrix changes during the stages of tissue repair, how matricellular proteins affect cell-extracellular matrix interactions, and how these proteins might be exploited for use therapeutically.