Method: Mononuclear cells were isolated from synovial fluid and peripheral blood samples of 25 children with JIA and age matched controls by Ficoll-Hypaque density centrifugation. Healthy donor monocytes, purified using Percoll discontinuous density gradients, were stimulated with TLR ligands with or without NGF addition. The expression of TrkA and p75NTR and the effects of TrkA activation or inhibition on TLR signalling and cytokine release were evaluated using real-time PCR, western blot and ELISA.
Result: We found that mononuclear cells of JIA patients from both peripheral blood and synovial fluid are characterized by a significant decrease in TrkA expression, while p75-NTR expression remains stable. In normal monocytes, phosphorylation of TrkA after NGF binding activated intracellular pathways that interfered with TLR signalling. Indeed NGF addition increased Akt phosphorylation, inactivated GSK3 and reduced IkB phosphorylation. Blocking TrkA enhanced inflammatory cytokine production, while reducing IL-10 synthesis and resulted in a greater activation of the NF-kB pathway and in inhibition of the PI3K pathway.
Conclusion: Our results imply that NGF is a relevant component of the endogenous mechanisms limiting excessive inflammatory response. Indeed, through TrkA NGF potentiates anti-inflammatory responses usually activated after TLR stimulation. The well-known increase in NGF in chronic arthritis patients may be de facto not efficacious in controlling inflammation because of the decrease of TrkA expression observed in JIA patients. This down-regulation of TrkA expression may influence the balance between inflammatory and anti-inflammatory pathways.
Disclosure: G. Minnone, None; G. Prencipe, None; R. Strippoli, None; L. De Pasquale, None; I. Caiello, None; F. De Benedetti, None; L. Bracci-Laudiero, None.