Kristen R. Taylor

Glycosaminoglycans and their proteoglycans: host-associated molecular patterns for initiation and modulation of inflammation.

Glycosaminoglycans, linear carbohydrates such as heparan sulfate and hyaluronan, participate in a variety of biological processes including cell‐matrix interactions and activation of chemokines, enzymes and growth factors. This review will discuss progress in immunology and the science of wound repair that has revealed the importance of glycosaminoglycans, and their proteoglycans, in the inflammatory process. Heparan sulfate enables growth factor function and modifies enzyme/inhibitor functions, such as antithrombin III and heparin cofactor II. Heparan sulfate also interacts with cytokines/chemokines and participates in leukocyte selectin binding to promote the recruitment of leukocytes. Chondroitin sulfate/ dermatan sulfate regulates growth factor activity and is an alternate modulator of heparin cofactor II. In addition, dermatan sulfate induces ICAM‐1 expression on endothelial cells and also recruits leukocytes via selectin interactions. Hyaluronan alternatively participates in leukocyte recruitment via interaction with CD44, while activating various inflammatory cells, such as macrophages, through CD44‐dependent signaling. Hyaluronan also signals through Toll‐like receptor 4 to induce dendritic cell maturation and promote cytokine release by dendritic cells and endothelial cells. Taken together, the field of glycosaminoglycan biology provides new clues and explanations of the process of inflammation and suggests new therapeutic approaches to human disease.—Taylor, K. R., Gallo, R. L. Glycosaminoglycans and their proteoglycans: host‐associated molecular patterns for initiation and modulation of inflammation. FASEB J. 20, 9–22 (2006)

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-β2 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.

NLRP3/Cryopyrin Is Necessary for Interleukin-1β (IL-1β) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation.

Patient Mutations in Doublecortin Define a Repeated Tubulin-binding Domain

Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. Here we show that the two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro andin vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a β-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif.

Cathelicidin Antimicrobial Peptides Block Dendritic Cell TLR4 Activation and Allergic Contact Sensitization

Cathelicidins are antimicrobial peptides of the innate immune system that establish an antimicrobial barrier at epithelial interfaces and have been proposed to have a proinflammatory function. We studied the role of cathelicidin in allergic contact dermatitis, a model requiring dendritic cells of the innate immune response and T cells of the adaptive immune response. Deletion of the murine cathelicidin gene Cnlp enhanced an allergic contact response, whereas local administration of cathelicidin before sensitization inhibited the allergic response. Cathelicidins inhibited TLR4 but not TLR2 mediated induction of dendritic cell maturation and cytokine release, and this inhibition was associated with an alteration of cell membrane function and structure. Further analysis in vivo connected these observations because inhibition of sensitization by exogenous cathelicidin was dependent on the presence of functional TLR4. These observations provide evidence that cathelicidin antimicrobial peptides mediate an anti-inflammatory response in part by their activity at the membrane.

Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS.

Fragments of hyaluronan released after injury bind and activate TLR4 in a complex with CD44. Here we investigated if the recognition of hyaluronan by CD44 and TLR4 alters lipopolysaccharide (LPS) responsiveness and thus could alter the septic response. In contrast to mice injected with LPS, mice exposed to hyaluronan prior to LPS had greatly decreased serum IL-6 and TNFalpha and were protected from symptoms of sepsis. The protective effect of HA was not seen in Cd44(-/-) mice. Consistent with our findings in vivo, addition of hyaluronan to macrophages before LPS exposure significantly decreased the release of IL-6 and TNFalpha and this effect was not seen in macrophages from Cd44(-/-) mice. Investigation of the mechanism responsible for inhibition of LPS activation showed hyaluronan treatment resulted in an increase in peritoneal macrophage A20 mRNA expression, and that this was significantly reduced in macrophages from Cd44(-/-) mice and Tlr4(-/-) mice. Suppression of the A20 response with siRNA inhibited the ability of hyaluronan to protect against the cytokine response to LPS. Therefore, our results show that hyaluronan acts through TLR4, CD44 and A20 to stimulate a unique cellular response that can protect against the septic response to LPS.

FGF-10 and specific structural elements of dermatan sulfate size and sulfation promote maximal keratinocyte migration and cellular proliferation

Fibroblast growth factor‐10 (FGF‐10) is essential for epithelial development, while other members of this family, such as FGF‐7, are not. FGF‐10 is abundantly released into wounds following injury, and likely an essential growth factor required for this process. To evaluate how activation of this growth factor is controlled, multiple glycosaminoglycans were combined with FGF‐10 assayed by measurement of the proliferation of cell lines expressing FGF receptor‐2‐IIIb, or keratinocyte migration in an in vitro wound repair assay. Dermatan sulfate (DS) exhibited greater potency than heparan sulfate or other chondroitin sulfates found in wounds. Structural variants of DS between 10 and 20 disaccharides containing iduronic acid showed maximal capacity to enable FGF‐10 receptor stimulation. Furthermore, FGF‐10 and DS markedly enhanced migration of keratinocytes in an in vitro wound scratch assay, while FGF‐7 or other glycosaminoglycans did not. These data strongly suggest that FGF‐10 activity is uniquely important in wound repair and that specific DS structural properties are necessary to promote FGF‐10 function. These observations identify a novel interplay between DS and FGF‐10 in mediating wound repair.