There is growing interest in the complex relationship between the nervous and immune systems and how its alteration can affect homeostasis and result in the development of inflammatory diseases. A key mediator in cross-talk between the two systems is nerve growth factor (NGF), which can influence both neuronal cell function and immune cell activity. The up-regulation of NGF described in inflamed tissues of many diseases can regulate innervation and neuronal activity of peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters, but can also directly influence innate and adaptive immune responses. Expression of the NGF receptors tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) is dynamically regulated in immune cells, suggesting a varying requirement for NGF depending on their state of differentiation and functional activity. NGF has a variety of effects that can be either pro-inflammatory or anti-inflammatory. This apparent contradiction can be explained by considering NGF as part of an endogenous mechanism that, while activating immune responses, also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression, such as that recently demonstrated in immune cells of arthritis patients, might prevent the activation by NGF of regulatory feed-back mechanisms, thus contributing to the development and maintenance of chronic inflammation.

Nerve growth factor (NGF) levels are highly increased in inflamed tissues, but their role is unclear. We show that NGF is part of a regulatory loop in monocytes: inflammatory stimuli, while activating a proinflammatory response through TLRs, upregulate the expression of the NGF receptor TrkA. In turn, NGF, by binding to TrkA, interferes with TLR responses. In TLR-activated monocytes, NGF reduces inflammatory cytokine production (IL-1β, TNF-α, IL-6, and IL-8) while inducing the release of anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist). NGF binding to TrkA affects TLR signaling, favoring pathways that mediate inhibition of inflammatory responses: it increases Akt phosphorylation, inhibits glycogen synthase kinase 3 activity, reduces IκB phosphorylation and p65 NF-κB translocation, and increases nuclear p50 NF-κB binding activity. Use of TrkA inhibitors in TLR-activated monocytes abolishes the effects of NGF on the activation of anti-inflammatory signaling pathways, thus increasing NF-κB pathway activation and inflammatory cytokine production while reducing IL-10 production. PBMC and mononuclear cells obtained from the synovial fluid of patients with juvenile idiopathic arthritis show marked downregulation of TrkA expression. In ex vivo experiments, the addition of NGF to LPS-activated juvenile idiopathic arthritis to both mononuclear cells from synovial fluid and PBMC fails to reduce the production of IL-6 that, in contrast, is observed in healthy donors. This suggests that defective TrkA expression may facilitate proinflammatory mechanisms, contributing to chronic tissue inflammation and damage. In conclusion, this study identifies a novel regulatory mechanism of inflammatory responses through NGF and its receptor TrkA, for which abnormality may have pathogenic implications for chronic inflammatory diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

ObjectiveTo identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammation.MethodsReal-time PCR, western blot and ELISA were performed to evaluate NGF, proNGF, their receptor and cytokine expression in synovial tissue and cells of patients with juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA), and controls.ResultsproNGF and not mNGF is the prevalent form measured in synovial fluids of patients with JIA and RA with synovial fibroblasts as a major source of proNGF in the inflamed synoviae. p75NTR, the specific receptor for proNGF, is the NGF receptor most expressed in mononuclear cells of patients with JIA, while TrkA is the prevalent receptor in healthy donors. In ex vivo experiments the effects of proNGF differ from those of mNGF, suggesting that the balance of p75NTR and TrkA expression represents a critical factor in regulating mNGF/proNGF functions, determining which intracellular pathways and biological activities are triggered. Contrary to NGF, proNGF administration increased inflammatory cytokines but not interleukin (IL)-10 expression, inducing a stronger activation of p38 and JNK pathways. proNGF effects depend on its binding to p75NTR, as inhibition of p75NTR with neutralising antibodies or LM11A-31 abolished proNGF-induced production of IL-6 in patients’ mononuclear cells, while inhibition of TrkA did not. There is a correlation in patients with arthritis between high p75NTR levels and severity of clinical symptoms.ConclusionsOur data suggest that an active proNGF-p75NTR axis promotes proinflammatory mechanisms contributing to chronic tissue inflammation, and that the use of p75NTR inhibitors may represent a new therapeutic approach in chronic arthritis.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1+, Rab5+) and recycling (Rab11+) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

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