Oligo form of hyaluronic acid suppresses TLR3-dependent cytokine expression in a TLR4-dependent manner

Fragments of Hyaluronan (HA) that are generated after injury have been proposed to be immunomodulatory and act as regulators of inflammation. Previous studies have found that some pro-inflammatory signals of injury are mediated by self-RNA via TLR3 activation. Conversely, HA fragments released after injury may be immunosuppressive, since addition of HA to macrophages before LPS (TLR4 ligand) significantly decreased IL-6, TNFalpha], and suppressed sepsis response in mice. In this study we investigated how HA may influence inflammation induced by TLR3 ligands. A pure, synthetic small MW form of HA (oligoHA) was added to MHS cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligoHA in co- treatments suppressed IL-6 and TNF[alpha] in comparison to poly(I:C) alone. IL-6 mRNA expression was also suppressed as measured by qPCR. To determine how oligoHA acts on macrophages: WT, Tlr2-/- or Tlr4-/- mouse-derived macrophages were similarly treated with oligoHA and poly(I :C). Tlr2-/- macrophages responded like wild-type (WT) cells and retained suppression of cytokines while Tlr4-/- macrophages did not. An increase in Traf1 (TLR negtative regulator) mRNA was observed after oligoHA treatment of WT but not in Tlr4-/- mouse macrophages, and oligoHA did not alter cytokine responsiveness in Traf1-/- macrophages. Therefore, our results show that oligoHA released after injury is an immunosuppressant that acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of innate immune signaling and suggests novel approaches for influencing autoimmune diseas