Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS
Introduction
Septic shock is characterized by tissue and organ damage resulting from high production of cytokines and low molecular weight mediators in response to large amounts of Gram-negative bacteria or purified LPS (Cook, 1998). LPS is a major component of the Gram-negative bacteria cell wall and is a potent stimulus for recognition by the host innate immune system. LPS binds to and signals through a receptor complex, which consists of TLR4, CD14, and MD2 (Kawai et al., 1999, Poltorak et al., 1998). Activation of TLR4 promotes NF-κB-mediated production of proinflammatory cytokines in many cell types (Akira and Takeda, 2004). Prior exposure to LPS both in vitro and in vivo can lead to desensitization of immune cells to subsequent challenge with LPS, a phenomenon that has been referred to as ‘endotoxin tolerance.’
Progress has been made in understanding mechanisms involved in the negative regulation of TLR signaling (Liew et al., 2005), an essential event through which the host attenuates TLR signaling to avoid detrimental effects of an excess inflammatory response. Several intracellular proteins including TNFα-induced protein 3 (TNFAIP3/A20) (Boone et al., 2004, Lee et al., 2000) have been identified as negative regulators of TLR signaling. A20-deficient mice develop severe inflammation and cachexia, are hypersensitive to both LPS and TNF, and die due to the failure to regulate TNF-induced NF-κB activation (Boone et al., 2004, Lee et al., 2000). Furthermore, the ability of heterologous A20 expression to inhibit LPS-induced NF-κB activity suggests that endogenous A20 may be important for the termination of LPS signals (Cooper et al., 1996). However, the endogenous molecules responsible for A20 activation and subsequent suppression of TLR4 activation remain unknown.
Recent observations have shown that fragments of hyaluronan (HA) can serve as an endogenous trigger of TLR signaling and are able to activate innate immune defense, promoting the production of cytokines by a variety of cell types (Jiang et al., 2005, Termeer et al., 2002). Normally, HA is a high molecular mass (∼107 Da) linear polysaccharide composed of repeating disaccharide units of β1–4 linked d-glucuronic acid (GlcA) and β1–3 linked N-acetyl-d-glucosamine (GlcNAc) residues (4GlcAβ1–3GlcNAc1β) (Kogan et al., 2007). HA is a major component of the extracellular matrix (Matsuno et al., 2008) and is thought to be involved in maintaining tissue hydration, the distribution and transport of plasma proteins, and in maintaining an intact matrix structure. Fragments of HA are generated during inflammation or injury through the activity of oxygen radicals or via enzymatic activity by hyaluronidase, β-glucuronidase, and hexosaminidase (Laurent et al., 1995). CD44, a receptor for HA, is involved in HA-induced cytokine release by forming a TLR4–CD44 complex (Taylor et al., 2007). In addition, there is an increasing body of evidence that CD44 is a negative regulator of TLR2- and TLR4-mediated inflammation (Kawana et al., 2008, Liang et al., 2007).
Some molecules that do not share structural homology with LPS can elicit a tolerant phenotype to LPS; a phenomenon that has been referred to as ‘cross-tolerance’ or ‘heterotolerance’. IL-1β, a mycoplasma lipopeptide (macrophage-activating lipopeptide-2; MALP-2) and lipoteichoic acid (LTA) have this ‘cross-tolerant’ capacity (Lehner et al., 2001, Medvedev et al., 2000, Sato et al., 2000). In this study, we show for the first time that HA and CD44 trigger alternate innate TLR4 signaling events to initiate a distinct response from LPS. These findings describe a previously unrecognized role for HA in protecting against unopposed LPS signaling.
Section snippets
Reagents, cells, cell lines, mice
The mouse alveolar macrophage cell line MH-S was purchased from American Type Culture Collection (ATCC, catalog CRL-2019). Cells were maintained in RPMI1640 media supplemented with l-glutamine, 10% heat-inactivated fetal calf serum (FCS), penicillin/streptomycin (100 units/ml and 50 mg/ml, respectively) and 50 μM 2-mercaptoethanol. HA was prepared by the method previously described (Taylor et al., 2004). Human umbilical cord HA was purchased from Sigma–Aldrich (St. Louis, MO) and contains at wide
Hyaluronan (HA) and CD44 protects mice from LPS-induced shock
HA binds a complex of CD44 and TLR4 on the cell surface to initiate signaling (Taylor et al., 2004, Taylor et al., 2007, Yamasaki et al., 2009). We employed a mouse model of LPS-induced endotoxic shock to examine whether the unique receptor complex occupied by HA could affect the response to LPS. C57BL/6J mice were first injected i.p. with endotoxin-free HA (20 mg/kg) or PBS. One hour after this pretreatment, a following challenge of LPS (20 mg/kg) or PBS was i.p. injected into each group and
Discussion
Septic shock is one of the most challenging problems in critical care medicine, and accounts for significant morbidity and mortality in medical and surgical intensive care units (Parrillo, 2008). In this current study we demonstrate that HA pretreatment protects mice from the LPS-induced endotoxic shock phenomenon and suppresses cytokine elevations in serum. These observations shed new light on potential elements that regulate the septic shock response, and may have important therapeutic
Conflict of interest
The authors state no conflict of interest.
Acknowledgements
We thank Drs. Shizuo Akira and Allen F. Ryan for the Myd88−/− and Trif−/− mice.
This work was supported by NIH grants P01HL057345, R01AR45676 and a VA Merit award R.L.G.
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