Journal: RMD Open

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

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: p75NTR, TrkA and sortilin expression levels in patients with JIA. (A) mRNA expression levels of TrkA, p75NTR and sortilin in freshly isolated mononuclear cells from peripheral blood of healthy children (CTRL PBMC) and in mononuclear cells from peripheral blood or from synovial fluids of patients with JIA (respectively, JIA PBMC and JIA SFMC) were quantified by real-time PCR analysis. JIA PBMC (n=34) and JIA SFMC (n=66) express low TrkA and high p75NTR mRNA levels compared with CTRL PBMC (n=10). Sortilin, a coreceptor for p75NTR essential for proNGF binding, is highly expressed in JIA SFMC (n=19) and expressed in JIA PBMC (n=13) and in CTRL PBMC (n=7). The results are expressed as arbitrary units (AU) after normalisation with the housekeeping gene GAPDH. (B) p75NTR mRNA levels are higher in SFMC than in PBMC of matched patients with JIA (n=18). (C) Sortilin mRNA levels are higher in SFMC than in PBMC of matched patients with JIA (n=11). (D) Western blot and densitometric analysis of three independent experiments confirmed that p75NTR was the prevalent NGF receptor in JIA mononuclear cells, while TrkA was the most expressed NGF receptor in CTRL PBMC. Sortilin is also highly expressed in JIA mononuclear cells. *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: RMD Open

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

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: Mature and immature NGF forms in patients with JIA and RA. (A,B) Only proNGF forms of different molecular weights but not mature NGF were detected by western blot in synovial fluids of 12 patients with JIA (40 μg total protein). (B) Blocking the anti-NGF antibody by adding mature NGF results in the disappearance of the specific proNGF bands observed in synovial fluids of three patients with JIA, as well as of the band of the 25 kDa commercial proNGF that was added as positive control. (C) In JIA and RA synovial fluids, proNGF and mature NGF (mNGF) concentrations were assessed using a newly developed ELISA showing that proNGF is 4.8-fold higher in JIA (n=27) and 16.8-fold higher in patients with RA (n=5) than mNGF. (D) Real-time PCR shows a very low expression of NGF mRNA in mononuclear cells obtained from peripheral blood of healthy donors (CTRL PBMC) or in mononuclear cells from peripheral blood (JIA PBMC) and from synovial fluids of patients with JIA (JIA SFMC). High NGF mRNA expression levels characterised synovial tissues (RA synovia; n=5). Fibroblast-like synoviocytes (FLS; n=3) of patients with RA express more NGF mRNA than FLS from osteoarthritis patients (OA FLS) (n=4) and control fibroblasts (CTRL FB; n=4). Results were expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (E) Both unstimulated (US) and LPS-stimulated JIA SFMC do not release proNGF or mNGF in the supernatants. Commercial proNGF and mNGF were added as positive controls. (F) Western blot shows the stability of exogenous added mNGF and proNGF. After its addition to culture media, proNGF is still detected after 3 hours and undergoes a minor maturation into mNGF in the first 2 hours of incubation (left side of F). mNGF is not degraded in the first 3 hours after its addition (right side of F). Commercial proNGF and mNGF were included as positive controls. (G) Neither proNGF nor mNGF were released in culture media of US or LPS-stimulated JIA SFMC after 18 hours of incubation. Exogenous proNGF or mNGF were detected only at time of supplementation (in agreement with F) but were no longer detectable after 18 hours of incubation. Commercial proNGF and mNGF were included as positive controls. (H) ELISA instead shows that conditioned media of RA FLS (n=5) have higher concentrations of proNGF than control fibroblasts (CTRL FB; n=5) and that the concentration of proNGF is higher than mNGF after 18 hours of incubation. *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood; RA rheumatoid arthritis; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Western Blot, Blocking Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Incubation, Concentration Assay, Isolation

Journal: RMD Open

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

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: Ex vivo effects of proNGF and NGF on JIA mononuclear cells. (A) No changes in cell viability were observed when mononuclear cells from synovial fluid (SFMC) of patients with JIA were treated for 24 hours with proNGF or mNGF, with or without LPS stimulation. Apoptosis was assessed by Annexin V flow cytometry analysis. The results represent one of three independent experiments performed in duplicate. (B) While proNGF addition alone did not induce the production of interleukin (IL)-6 in unstimulated SFMC, in LPS-activated cells proNGF induced IL-6 release in a dose-dependent manner with a maximal effect at 200 ng/mL (n=4). (C) proNGF stimulation significantly increased IL-6, IL-1β, IL-8 and tumor necrosis factor-α (TNF-α) mRNA expression levels in LPS-stimulated SFMC (n=18) of patients with JIA after 3 hours of incubation, but did not modify IL-10 mRNA expression in either CTRL or JIA mononuclear cells. Mature NGF treatment did not significantly modify proinflammatory cytokine levels or IL-10 mRNA expression in JIA mononuclear cells, while it increased IL-10 mRNA levels in CTRL PBMC. Results are expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene GAPDH. (D) proNGF treatment significantly increased IL-6 release after 18 hours (measured by ELISA) in LPS-stimulated SFMC from patients with JIA (n=16) compared with PBMC of healthy CTRL (n=6). Mature NGF treatment did not affect IL-6 protein levels. *p<0.05. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Ex Vivo, Flow Cytometry, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Isolation

Journal: RMD Open

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

doi: 10.1136/rmdopen-2017-000441

Figure Lengend Snippet: proNGF and NGF activated different intracellular pathways. (A) The neutralisation of p75NTR using either a specific anti-p75NTR antibody (a-p75; 2.5 µg/mL) or using the specific pharmacological inhibitor LM11A.31 (10 nM), decreased interleukin (IL)-6 release in LPS-activated SFMC of patients with JIA (n=16) treated with proNGF. On the contrary, the blocking of TrkA with a neutralising antibody (a-TrkA; 3 µg/mL) did not affect IL-6 release. (B) Western blot shows an increased phosphorylation of p38 and JNK after 5 minutes of proNGF stimulation in LPS-treated SFMC. mNGF treatment did not affect the phosphorylation of these downstream molecules. The result is representative of one out of three independent experiments. Results of the densitometric analysis of all experiments are expressed as arbitrary units (AU). (C) In LPS-activated SFMC of three different patients with JIA, preincubation with the inhibitor LM11A-31 (10 nM), which blocks the binding of proNGF to p75NTR, abolished the phosphorylation of p38 and JNK induced after 5 minutes of proNGF addition. Results of the densitometric analysis of all experiments are expressed as arbitrary units (AU). *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide ; NGF, nerve growth factor; SFMC, mononuclear cells isolated from synovial fluids.

Article Snippet: Mononuclear cells were treated with or without mNGF or mutated cleavage-resistant proNGF (Alomone Labs, Jerusalem, Israel) at a concentration, respectively, of 100 ng/mL and 200 ng/mL, calculated to yield the same molar concentration as described in neurons.

Techniques: Blocking Assay, Western Blot, Binding Assay, Isolation