Nerve Growth Factor Downregulates Inflammatory Response in Human Monocytes through TrkA

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.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, thu

ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis

OBJECTIVE: To identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammation. METHODS: Real-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. RESULTS: proNGF 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. CONCLUSIONS: Our 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

Different responses of PC12 cells to different pro-nerve growth factor protein variants

The present work aimed to explore the innovative hypothesis that different transcript/protein variants of a pro-neurotrophin may generate different biological outcomes in a cellular system. Nerve growth factor (NGF) is important in the development and progression of neurodegenerative and cancer conditions. Mature NGF (mNGF) originates from a precursor, proNGF, produced in mouse in two major variants, proNGF-A and proNGF-B. Different receptors bind mNGF and proNGF, generating neurotrophic or neurotoxic outcomes. It is known that dysregulation in the proNGF/mNGF ratio and in NGF-receptors expression affects brain homeostasis. To date, however, the specific roles of the two major proNGF variants remain unexplored. Here we attempted a first characterization of the possible differential effects of proNGF-A and proNGF-B on viability, differentiation and endogenous ngf gene expression in the PC12 cell line. We also investigated the differential involvement of NGF receptors in the actions of proNGF. We found that native mouse mNGF, proNGF-A and proNGF-B elicited different effects on PC12 cell survival and differentiation. Only mNGF and proNGF-A promoted neurotrophic responses when all NGF receptors are exposed at the cell surface. Tropomyosine receptor kinase A (TrkA) blockade inhibited cell differentiation, regardless of which NGF was added to culture media. Only proNGF-A exerted a pro-survival effect when TrkA was inhibited. Conversely, proNGF-B exerted differentiative effects when the p75 neurotrophin receptor (p75NTR) was antagonized. Stimulation with NGF variants differentially regulated the autocrine production of distinct proNgf mRNA. Overall, our findings suggest that mNGF and proNGF-A may elicit similar neurotrophic effects, not necessarily linked to activation of the same NGF-receptor, while the action of proNGF-B may be determined by the NGF-receptors balance. Thus, the proposed involvement of proNGF/NGF on the development and progression of neurodegenerative and tumor conditions may depend on the NGF-receptors balance, on specific NGF trancript expression and on the proNGF protein variant ratio

Monocytes and macrophages as biomarkers for the diagnosis of megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells

Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

We have recently provided new evidence for a role of p75NTR receptor and its preferential ligand proNGF in amplifying inflammatory responses in synovial mononuclear cells of chronic arthritis patients. In the present study, to better investigate how activation of the p75NTR/proNGF axis impacts synovial inflammation, we have studied the effects of proNGF on fibroblast-like synoviocytes (FLS), which play a central role in modulating local immune responses and in activating pro-inflammatory pathways. Using single cell RNA sequencing in synovial tissues from active and treatment-naïve rheumatoid arthritis (RA) patients, we demonstrated that p75NTR and sortilin, which form a high affinity receptor complex for proNGF, are highly expressed in PRG4pos lining and THY1posCOL1A1pos sublining fibroblast clusters in RA synovia but decreased in RA patients in sustained clinical remission. In ex vivo experiments we found that FLS from rheumatoid arthritis patients (RA-FLS) retained in vitro a markedly higher expression of p75NTR and sortilin than FLS from osteoarthritis patients (OA-FLS). Inflammatory stimuli further up-regulated p75NTR expression and induced endogenous production of proNGF in RA-FLS, leading to an autocrine activation of the proNGF/p75NTR pathway that results in an increased release of pro-inflammatory cytokines. Our data on the inhibition of p75NTR receptor, which reduced the release of IL-1β, IL-6 and TNF-α, further confirmed the key role of p75NTR activation in regulating inflammatory cytokine production. In a set of ex vivo experiments, we used RA-FLS and cultured them in the presence of synovial fluids obtained from arthritis patients that, as we demonstrated, are characterized by a high concentration of proNGF. Our data show that the high levels of proNGF present in inflamed synovial fluids induced pro-inflammatory cytokine production by RA-FLS. The blocking of NGF binding to p75NTR using specific inhibitors led instead to the disruption of this pro-inflammatory loop, reducing activation of the p38 and JNK intracellular pathways and decreasing inflammatory cytokine production. Overall, our data demonstrate that an active proNGF/p75NTR axis promotes pro-inflammatory responses in synovial fibroblasts, thereby contributing to chronic synovial inflammation, and point to the possible use of p75NTR inhibitors as a novel therapeutic approach in chronic arthritis