Presentation: Induction of Wnt Expression by Innate Immune Mechanisms in Inflamed Synovium (2007)

137 Induction of Wnt Expression by Innate Immune Mechanisms in Inflamed Synovium

Objective:
The development of rheumatoid arthritis (RA) is associated with hyperplasia and inflammation of the synovium. Mechanisms whereby inflammation stimulates aberrant growth and pathologic changes in the pannus are under intense investigation. During embryogenesis both the Wnt-Frizzled and Toll-like receptors (TLR) systems control embryonic tissue growth and patterning. Hence, we evaluated the interaction of the innate immune system to cross modulate expression of wnt growth factors in adult inflammation.
Methods
Murine knee synovium was dissected and dissociated in collagenase and synovial fibroblasts were cultured. Fibroblast-like synoviocytes (FLS) were plated in 6 well plates in media with 0.1% FBS overnight. The cells were treated with TNFa, IL-1b and TLR ligands and RNA isolated. Adult C57Bl/6, TLR-2-/-, TLR-4-/-, Toll-interleukin 1 receptor domain-containing adapter protein (TIRAP)-/- and MyD88-/- mice were injected 150ul of pooled K/BxN serum i.p. on Day 0. Mice were sacrificed at the indicated time points and the wrists were snap frozen in liquid nitrogen for mRNA analysis. Wnt mRNA expression was examined by quantitative PCR.
Results
Wild type FLS showed increased expression of specific wnt mRNA with IL-1b, TNFa and lipopolysaccharide (LPS) treatment. Wnt 5a mRNA levels increased 5.7, 3.8 and 2.1 fold; and Wnt 10b mRNA levels increased 9.8, 5.8 and 3.1 fold respectively. The peak wnt mRNA expression levels in FLS was after 8 hr of IL-1 and 24hr of TNFa treatment. FLS were treated with a panel of known TLR ligands. Amongst these the TLR-2 and 4 agonists showed the greatest induction of Wnt10b gene expression, namely FLS1 (6.1 fold), Pam3CSK4 (5.2 fold) and LPS (3.1 fold). To assess the in vivo joint expression of wnt ligands a time course experiment using the K/BxN serum transfer models showed peak expression occurred rapidly by Day 1 in inflamed paws. K/BXN serum was then transferred to mice deficient in TLR-2, -4, TIRAP and MyD88. Paw expression of Wnt-5a and Wnt-10b mRNA increased in TLR-2-/- and TLR-4-/- mice similar to wild type mice; however expression levels did not change in MyD88-/- and TIRAP-/- mice.
ConclusionFLS expression levels of Wnt-5a and Wnt-10b were augmented after exposure to proinflammatory cytokines TNFa and IL-1b, albeit with different kinetics. Treatment of FLS with TLR-ligands similarly increased expression of Wnt genes, particularly TLR-2 and -4 ligands. In a murine arthritis model, both TLR-2 and -4 may have redundantly contributed to increased expression of wnt genes. Deficiencies in the shared signaling intermediates TIRAP and MyD88 abrogated the increase in ligand expression, indicating that TLR signaling contributes to inflammation associated wnt gene expression.

 J. Kim, None; N. Flores, None; E. Grauel, None; S. Gardne, None; M. Corr, None.