PC1/3 protein expression and down-regulation by shRNA in NR8383 cells.

<p>A. qPCR analysis of the relative expression of PC1/3 in NR8383 cells stably transduced with non-target (NT) shRNA and PC1/3 shRNA. Relative expression was normalized on β-actin (<i>n</i> = 3). B–C. Western blot of PC1/3 in NR8383 cells stably transduced with NT shRNA or PC1/3 shRNA as well as AtT-20 cells using a PC1/3 antibody targeted against the C-terminal domain (B) or the catalytic domain (C). 15 µg of AtT-20 protein and 50 µg of NR8383 cells were loaded. Pro PC1/3 is detected around 90 kDa, full-length PC1/3 (PC1/3) is detected at 87 kDa and fully C-terminally matured PC1/3 (ΔCT PC1/3) is detected at 66 kDa. C. Confocal microscopy of PC1/3 by indirect immunofluorescence on NR8383 cells expressing PC1/3 shRNA (b) and NT shRNA (a) using anti-PC1/3 directed against the C-terminal domain. A positive control (AtT-20) is shown, which exhibits specific secretory granule-like labeling (c) (3× magnification in the rectangle inset). d- is a Z-stack reconstruction of confocal plane from (c) showing TGN labeling of PC1/3 in AtT-20 cells.</p

Effects of PC1/3 down-regulation on vesicle trafficking markers protein expression.

<p>Representative western blots (20 µg of protein) and gels optic density (OD) quantification showing the relative levels of vesicle trafficking markers EEA1 (A), RAB5 (B), RAB8 (C), RAB7 (D), RAB9 (E) and RAB11 (F) between NR8383 cells expressing control shRNA (NT) and those expressing shRNA directed against PC1/3 (SH). The actin loading control is included. * = 0.05 ** = 0.001 ***<0.0001 p-values, Student’s t-test n = 5–8.</p

Effects of PC1/3 down-regulation on cytokine secretion in NR8383 cells.

<p>Cytokine secretion measured by ELISA in control NR8383 cells expressing control shRNA (NT) and shRNA directed against PC1/3 in culture media with or without LPS stimulation (100 ng/mL) at various time points. A. TNF-alpha 4 h, B. TNF-alpha 24 h, C. IL-1beta 4 h, D. IL-1beta 24 h and E. IL-6 24 h. p-values were obtained using Student’s t-test (n = 4–6).</p

Proprotein Convertase 1/3 (PC1/3) in the Rat Alveolar Macrophage Cell Line NR8383: Localization, Trafficking and Effects on Cytokine Secretion

<div><p></p><p>The proprotein convertase 1/3 (PC1/3) is an important post-translational processing enzyme for the activation of precursor proteins within the regulated secretory pathway. Well characterized for its role in the neural and endocrine systems, we recently reported an unconventional role of PC1/3 as a modulator of the Toll-like receptor innate immune response. There are only a few reports that have studied PC1/3 expression in macrophages, and more investigation is needed to better characterize its function. These studies would greatly benefit from model cell lines. Our study aims to identify and characterize PC1/3 in a relevant model macrophage cell line and to determine the links between PC1/3 and innate immune cellular responses. We describe the rat alveolar cell line, NR8383, as expressing PC1/3 and the most common Toll-like receptors. In NR8383 cells, PC1/3 is localized at the Trans-Golgi network and traffics to lysosome related vesicles upon lipopolysaccharide stimulation. Moreover, we report the co-localization of PC1/3 and Toll-like receptor 4 upon lipopolysaccharide stimulation. Down regulation of PC1/3 by shRNA produce a similar phenotype in NR8383 to what we previously reported in isolated peritoneal macrophages. PC1/3 shRNA induced changes in the cellular organization and expression of the specific trafficking regulator RAB GTPase. As a consequence, NR8383 down-regulated for PC1/3, present an abnormal cytokine secretion profile. We conclude that the NR8383 cell line represents a good model to study PC1/3 in macrophages and we present PC1/3 as an important regulator of vesicle trafficking and secretion in macrophages.</p></div

LPS induces PC1/3 trafficking and co-localization with TLR4.

<p>Confocal images of NR8383 cells double-labeled with anti-PC1/3 (green) and anti-TLR4 (red) and incubated with 100 ng/ml LPS for the indicated time. Insets represent 3× magnification of regions where PC1/3 and TLR4 show partial co-localization.</p

Characterization of the NR8383 cell line.

<p>A. Expression of TLR in NR8383 by RT-PCR. B. Expression of PCs in NR8383 by RT-PCR with the expected band length in base pairs (bp). C. Expression of proSAAS and PC1/3 by qRT-PCR. Negative control (CTRL –) is a non template control and positive control (CTRL +) is 10 pg of rat proSAAS cloned into pCDNA3.1 vector. D. Northern blot analysis of PC1/3 in NR8383, PMA-induced THP-1 and control β-TC3.</p

Effects of PC1/3 down-regulation on vesicle trafficking markers.

<p>Confocal images of NR8383 cells stably transformed with control shRNA (NT) and shRNA directed against PC1/3 labeled with (A) early endosome marker EEA1 or RAB5. (D) baso-lateral membrane transport marker RAB8 and (F) late endosome/lysosome marker RAB7. In (A), arrows indicate punctate labeling near the plasma membrane. Insets represent 3× magnification. (B–C, G) EEA1 (B), RAB5 (C) and RAB7 (G) labelled vesicles area was estimated using ImageJ software. Vesicles where considered as ovals and area was calculated using the following formula: π×a×b where a and b are the two largest diameters. E. The ratio between peripheral and total integrated intensity of RAB8 labeling is represented. H. RAB7 peri nuclear integrated intensity is represented. * = 0.05 ** = 0.001 ***<0.0001, p-values, Student’s t-test. Quantification was performed on randomly selected field of view on two independent shRNA cell lines n = 4–6.</p

Mutation in the spike glycoprotein of mutant virus delays viral spreading compared to the reference strain in mixed primary CNS cultures from BALB/c mice.

<p>Mixed primary cultures from BALB/c mice brain were infected with rOC/ATCC or rOC/S_G758R at MOI 0.03. Viral spread was evaluated at 8, 24 and 48 hpi. Neurons were stained in green with a mAb against microtubule-associated protein 2 (MAP2) antibody and the S viral glycoprotein in red, was detected with a rabbit antiserum. Results are representative of three independent experiments. Magnification 200x.</p

Both variants rOC/ATCC and rOC/S_G758R are neuroinvasive and neurovirulent in 10 day-old BALB/c mice infected by the intranasal route.

<p>10-day old BALB/c mice received 10^3.25 TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R or PBS by the IN route. (A) Survival curves of mice in percentage (%) during 21 days post-infection. Difference between the two variants was significant (** P≤0.01) (B) Surviving BALB/c mice were weighed every 2 days after infection during 21 dpi to estimate weight variations. The weight variation was expressed in %, compared to day 0, which was set at 100%. Production of infectious viral particles was measured in brains (C) and spinal cords (D) every 2 days for 21 dpi. LOD represents the Limit of Detection of infectious viral particles. Results shown are the mean values (with standard deviations) of three independent experiments.</p

A delay in viral spread is observed in brains of rOC/S_G758R -infected mice compared to rOC/ATCC after intracerebral infection in 21 day-old BALB/c female mice.

<p>Histological examination of virus spread within the brain. 21 day-old BALB/c mice received 10^2.5TCID₅₀/10μL of rOC/ATCC or rOC/S_G758R, or PBS by the IC route. Detection of viral antigens in the olfactory bulb (A) or in the hippocampus (B) of infected mice at 5 and 7 dpi at magnitude X40. Black arrows indicate viral particles staining for the S protein.</p