Modulation of Nerve Growth Factor receptors in human monocytes and their influence in pulmonary inflammatory diseases

Neurotrophins (NTs) are a family of growth/survival factors with well-established functions in the nervous system. In the last decade, novel biological actions, from oncogenicity to inflammation, have been attributed to these factors. In particular, Nerve Growth Factor (NGF) acts through two different classes of receptors: the high affinity transducing TrKA receptor, associated to proliferation/survival, and the low affinity p75 receptor that, depending on its cross talk with TrKA, induces either apoptosis or survival. Recently, NGF and its receptors have been detected in healthy pulmonary tissues and seem to be involved in pulmonary inflammatory diseases. Since the expression of these NGF receptors in circulating monocytes is controversial, our initial aim was to investigate the role of these receptors both in pulmonary tissues and in peripheral blood monocytes of patients with Chronic Obstructive Pulmonary Disease (COPD). We therefore analyzed 38 healthy control subjects divided in two groups based on their smoking status. Serendipitously, the data obtained in these two control groups may have a value of their own, with potential implications in preventive medicine. We show here that in healthy subjects, smoking induces an early increase in p75 expression in monocytes, while TrKA seems unaffected. Furthermore, our control subjects could be divided in three subsets according to the constitutive TrKA expression in monocytes: TrKA-negatives, -intermediates (up to 50%) or -high (> 50%), independently on their smoking status. Since TrKA activation promotes inflammation, we hypothesize that subjects with high-TrKA monocytes could be more prone to pulmonary inflammatory diseases, such as COPD. In fact, all 28 COPD patients in our series belonged to the high-TrKA subset, while all long term heavy smokers with no evidence of pneumologic diseases belonged to TrKA-negative subset. In conclusion, our data support the hypothesis that, since TrKA expression promotes survival, elevated levels of TrKA-positive monocytes may render subjects more prone to long term inflammatory diseases (e.g., COPD). Moreover, in patients constitutively expressing high levels of TrKA in monocytes, the smoking-dependent increase in p75 may in turn further extend monocytes survival, contributing to a chronic inflammation. Furthermore, the early increase in p75 expression in monocytes following smoking may support the hypothesis that p75 determination might represent a novel marker for passive smoking. Acknowledgments. This study was totally supported by Fondazione Cassa di Risparmio di Tern

Nicotine induces overexpression of low affinity p75 NGF receptor in monocytes

In inflammatory pulmonary diseases, cigarette smoking is a major risk factor influencing the phenotype of immune cells and their functions (Arnson et al., 2010). Nicotine is a relevant constituent of cigarette smoking and, on monocytes, it binds nicotinic acetylcholine receptors inducing a quantitative increase in the levels of CD14 and a decrease in TNF-a. Altogether, these data supports an anti-inflammatory effect of nicotine on these cells.Our previous studies have shown that NGF and its receptors TrKA and p75 are involved in inflammatory pulmonary diseases. In particular, we have shown that smokers present an increase in p75 expression on monocytes, but the cause that triggers this increase is actually unknown. In addition, cigarette smoking seems not to vary the TrKA expression on these cells. The aim of our study was to investigate in vitro the biological effects of nicotine on the percentage of TrKA- and p75-positive monocytes from human healthy non smoker donors. Cytofluorimetric investigation confirms that in monocytes nicotine treatment does not influence the percentage of TrKA, but induces a relevant dose-dependent increase in the percentage of p75 that, in turn, could be an element in the general anti-inflammatory effects of nicotine. Consequently, in order to investigate the involvement of p75 in inflammatory/non-inflammatory mechanisms, we added in vitro the inflammatory stimulus MCP-1 on monocytes. Cytofluorimetric investigation have shown that, in this latest inflammatory condition, the percentage of p75-positive monocytes was greatly reduced. In conclusion, our data support the hypothesis that nicotine-induced p75 overexpression could be involved in its anti-inflammatory effect

A Novel Role for Tm7sf2 Gene in Regulating TNFα Expression

<div><p>We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2^+/+ and Tm7sf2^−/− mice, to determine the <i>in vitro</i> effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an <i>in vivo</i> model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2^−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.</p></div

Tm7sf2 gene presides over an anti inflammatory loop.

<p>(a) Nuclear translocation of Nrf2 and NF-κB in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin. At the indicated times, cells were collected and nuclear extracts subjected to Western blotting analyses with the indicated antibodies. Lamin B was used as loading control; (b) TNFα and HO-1 gene expression in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. (c) TNFα expression in KO MEFs grown in 10%FBS and treated with 1 µM thapsigargin for 6 hr and subjected to real time PCR analysis. Expression of each gene was normalized to GAPDH and reported as 2^−ΔΔCt. Relative mRNA level of WT untreated cells was assumed as 1. Results are given as mean ± s.d., (n = 7). *p<0.05 vs. FBS grown WT MEFs, # p<0.05 vs. the respective WT; NF-κB activation by Western blotting in (d) MEFs, grown in DMEM plus 10%FBS and treated with 1 µM thapsigargin for 6 hr, and (e) KO MEFs transfected with hTm7sf2 gene, incubated for 24 hr in culture medium containing 5% LPDS, and treated with 1 µM thapsigargin for 6 hr (n = 3); (f) Ear oedema induction in WT and KO mice. Mice were treated with 4 nmol TPA on both sides of the left ear. Ear thickness was measured at the indicated time points with a digital calliper. Values represent mean ± s.d. (n = 8). *p<0.05 vs. control WT, # p<0.05 vs. the respective WT. Representative histological sections of ear pinnae 24 hr after ear oedema induction. The sections were stained with H-E. Images magnification, ×100.(g) TNFα gene expression in WT and KO mice. Mice, i.p. injected with 1 mg/Kg tunicamycin, were sacrificed at 48 hr and kidneys used for real time PCR analysis. Expression of the gene was normalized to GAPDH and reported as 2^−ΔΔCt . Relative mRNA level of WT untreated mice kidney was assumed as 1. Results are given as mean ± s.d., (n = 7). *p<0.05 vs. control WT, # p<0.05 vs. the respective WT.</p

Tm7sf2 gene regulates LXR activation.

<p>(a) Ear oedema induction in WT and KO mice in the presence of T0901317. Mice were treated with 4 nmol TPA on both sides of the left ear and with 10 mM T0901317 at 45 min and 4 hr after TPA application. Ear thickness was measured after 24 hr with a digital calliper. Values represent mean ± s.d., (n = 8). *p<0.05 vs. control WT, # p<0.05 vs. the respective WT. § p<0.05 vs. the respective TPA-treated ear. Representative histological sections of ear pinnae 24 hr after ear oedema induction. The sections were stained with H-E. Images magnification, ×100. (b) NF-κB activation and (c) TNFα expression in MEFs grown in DMEM plus 5% LPDS, pre-treated for 1 hr with increasing concentrations of T0901317, then treated with 1 µM thapsigargin for 6 hr, and subjected to Western blotting and real time PCR analyses. Expression of each gene was normalized to GAPDH and reported as 2^−ΔΔCt. Relative mRNA level of WT untreated cells was assumed as 1. Results are given as mean ± s.d., (n  = 4). *p<0.05 vs. FBS grown WT MEFs, # p<0.05 vs. the respective WT.</p

Electron microscopy analysis of autophagosome.

<p>(a) Transmission electron microscopic analysis of WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 24 hr, A: autophagosome, N: nucleus; (b) Conversion of LC3I in LC3II during ER stress-induced autophagy by Western blotting.</p

Tm7sf2 gene controls eIF2α activation and attenuates ATF4 protein levels.

<p>(a) ER stress response in WT and KO MEFs. MEFs, grown in DMEM plus 5%LPDS, were treated with 1 µM thapsigargin for 18 hr, lysed and analyzed by Western blotting with the indicated antibody. β-actin and eIF2α were used as loading controls. (b) Time-course of eIF2α phosphorylation. WT and KO MEFs, treated for the indicated time with 1 µM thapsigargin, were lysed and analyzed for eIF2α phosphorylation. β-actin and eIF2α were used as loading controls. (c) ER stress response activation in mice. Kidneys of WT and KO mice, i.p. injected with 1 mg/Kg tunicamycin and sacrificed at 48 hr, were analyzed by Western blotting with the indicated antibody. β-actin was used as loading control.</p

The lack of Tm7sf2 gene inhibits the increase in hepatic cholesterol levels.

<p>(a) Mouse weight changes in response to tunicamycin administration. WT and KO mice were i.p. injected with 1 mg/Kg tunicamycin and mouse weight recorded at the beginning and the end of the treatment and reported as difference between the two values for each mouse. (b) Hepatic cholesterol levels of WT and KO mice by TLC analysis. (100% = 2.45 mg/g tissue). (c) Conversion of LC3I in LC3II during TN-induced autophagy in WT and KO mice kidney by Western blotting. Data represent mean ± s.d. of n = 7 mice. *p<0.05 vs. control WT, # p<0.05 vs. the respective WT.</p