36 research outputs found

    Early-age-related changes in proteostasis induce inflammation.

    No full text
    <p>The total protein lung- (A) and liver- (B) lysates from control, CLP or i.t. (1 µg/gm bw) <i>Pa</i>-LPS induced murine model (n = 3) were immunoblotted for Ub (ubiquitin), PSMB6 (proteasome-subunit), VCP (UPR), NFκB (Inflammation), p-eIF2α (protective stress response) and β-actin. (<b>A</b>) The adult mice lungs show constitutive increase in NFκB and p-eIF2α levels and accumulation of ubiquitinated-proteins while levels of proteasomal subunit, PSMB6 decrease. The <i>Pa</i>-LPS further induces the levels of these proteins and VCP in adult mice while PSMB6 is lower indicating that proteostasis-imbalance in adult subjects is a critical early-age-related change that may induce immunopathogenesis of chronic- of fatal- injury in older subjects. (<b>B</b>) The liver shows a constitutive increase in VCP expression in the adult mice which correlates with higher accumulation of ubiquitinated proteins. The bottom panel (A & B) shows the densitometric analysis of VCP expression in both lung and liver normalized to β-actin (Mean ± SEM, n = 3, *p<0.05). (<b>C & D</b>) Of note here, both lung (C) and liver (D) also show accumulation of ubiquitinated-proteins in the insoluble protein fraction of adult mice as compared to the pediatric (n = 3) but lungs have relatively less accumulation of ubiquitinated proteins in insoluble fraction as compared to the liver. <i>The early-age-related proteostasis-imbalance induces NFκB mediated inflammatory response</i>.</p

    Salubrinal controls CLP induced inflammatory response.

    No full text
    <p>The peritoneal lavage from age- and sex- matched control, CLP and/or intraperitoneal (i.p.) salubrinal (1 mg/kg/bw) groups of C57BL6 mice (n = 3–4) were used to quantify changes in IL-6 (A) levels, and number and pro-inflammatory state of inflammatory cells (B). (<b>A</b>) Salubrinal is effective in significantly (p = 0.05) controlling the CLP induced pro-inflammatory cytokine, IL-6 levels. (<b>B</b>) The flow cytometry analysis shows the number (x-axis) of peritoneal neutrophils (NIMP-R14), macrophages (Mac3) and CD4+ T cells (CD4) isolated from control- and CLP- groups after 24 hrs, with- and without- salubrinal treatment. The pro-inflammatory state of individual cell population was verified by co-staining for intracellular IFNγ (y-axis). Data shows increase in numbers of neutrophils (upper panel) and macrophages (middle panel) with decreasing IFNγ levels after CLP while number of CD4<sup>+</sup> T cells (lower panel) decrease and have increased IFNγ levels. <i>Salubrinal is effective in controlling CLP induced inflammatory response</i>.</p

    Early-age-related changes in VCP expression correlates with ubiquitin accumulation and hyper-inflammatory response.

    No full text
    <p>The longitudinal lungs sections from control, CLP or i.t. <i>Pa</i>-LPS (1 µg/gm bw) induced murine model (n = 3–4) were processed for histological evaluation. (<b>A</b>) We verified by TUNEL assay that accumulation of damaged- or misfolded- proteins leads to an increase in lung cell apoptosis in the adult mice by quantifying the changes in number of TUNEL positive cells. This is further exacerbated by <i>Pa</i>-LPS- or CLP- induced injury, verifying the correlation of ubiquitinated protein accumulation to increased apoptosis. The bottom panel of each staining shows the spearman's correlation coefficient analysis of the number of apoptotic cells in pediatric- and adult- mice (10 uniform representative areas from each mouse). (<b>B</b>) The adult mice show a constitutive increase in inflammation as compared to the pediatric group (H&E) that correlates with an increase in NFκB (C) and VCP (D) protein levels. The right panel of each staining shows the spearman's correlation coefficient analysis of fluorescence intensity in pediatric- and adult- mice (10 uniform representative areas from each mouse). (<b>E</b>) The <i>Pa</i>-LPS and CLP treatments induce the accumulation of ubiquitinated proteins that correlates with further increase in protein levels of NFκB (C) and VCP (D). <i>Data confirms that early-age-related proteostasis-imbalance augments Pa-LPS or sepsis induced injury</i>.</p

    Modulating CFTR expression may limit SHS induced bacterial survival.

    No full text
    <p>(A) Immunoblotting of total protein extracts from RAW264.7 cells treated with VRT-532 (10μM; overnight, a known CFTR corrector and potentiator) or untreated group (control), show slightly higher protein levels of CFTR and reduced NF-κB levels in the treatment group as compared to the controls. β-actin was used as a loading control (n = 3). (B) CFTR and NF- κB protein expression (in 3A) was normalized to β—actin using an Image-J software. The densitometry analysis verifies that VRT-532 treatment can decrease NF-κB protein expression (*p<0.05) as compared to untreated control. (C) RAW264.7 cells were seeded on a 24-well plate and treated overnight with VRT-532 (10μM). Next, these cells were infected with <i>PA01</i>-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150mins. Representative bright field (top) and fluorescent microscopy images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (D) CSE treatment (in 3C) significantly (**p<0.01) inhibits bacterial phagocytosis, while VRT-532 is unable to restore SHS impaired phagocytosis. (E) In a separate experiment (as described in 3C), media (100μl) was collected, spread on agar plates, and then incubated overnight at 37°C. CFU counts of the extracellular bacteria (media) indicates that VRT-532 treatment can significantly (*p<0.05) decrease SHS induced bacterial survival. Note, this experiment was done in parallel with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121200#pone.0121200.g002" target="_blank">Fig. 2E</a> (left panel, Rutin Hydrate), hence control and CSE samples used are common.</p

    Transient transfection with WT-CFTR rescues SHS impaired bacterial phagocytosis and limits survival.

    No full text
    <p>(A) RAW264.7 cells were seeded on a 24-well plate and transiently transfected (using Lipofectamine) with pcDNA3.1 control vector or pcDNA3.1-WTCFTR for 48 hours. Cells were infected with <i>PA01</i>-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150 min before microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (B) Quantification of bacterial phagocytosis (from 4A) shows that transfection with WT-CFTR significantly (*p<0.03) rescues SHS impaired phagocytosis. (C) RAW264.7 cells were treated (as described in 4A) and media (100 μL) was collected and spread on 2% LB agar plates. The plates were incubated overnight at 37°C followed by quantification of bacterial survival by colony forming unit (CFU) counts. Transient transfection with WT-CFTR significantly (*p<0.02) limits bacterial survival in the CSE/SHS treated group, but bacterial survival remains higher than the untreated controls suggesting higher CFTR expression may be needed to limit bacterial burden.</p

    Early-age-related proteostasis-imbalance is critical for immunopathogenesis of chronic or fatal disease.

    No full text
    <p>Our data suggest that early-age-dependent decrease in proteasomal activity (PSMB6) results in accumulation of damaged- or misfolded- ubiquitinated proteins leading to an increase in VCP activity as a cytoprotective ER stress response. The sustained increase in VCP expression augments NFκB activation that mediates chronic inflammatory- and apoptotic- signaling. Moreover, accumulation of ubiquitinated proteins has a synergistic effect on these detrimental processes, leading to chronic or potentially fatal injury. Our data suggest the therapeutic potential of salubrinal and selective VCP inhibition in controlling the accumulation of ubiquitinated proteins (proteostasis-imbalance) and NFκB mediated chronic or fatal disease. <i>We identify here a promising therapeutic strategy to restrain the immunopathogenesis of chronic or fatal injury in older subjects</i>.</p

    Proteasome inhibition regulates protein turnover rates.

    No full text
    <p>The human bronchial epithelial cells (HBE) were treated for 2 hours (2 h, partial) or overnight (O/N, broad) with low dose proteasome inhibitor (MG-132, 1 µM) and accumulation of newly synthesized ubiquitinated protein was monitored over time by metabolic labeling and immunoprecipitation. Lower levels of ubiquitinated proteins in overnight MG132 treated cells during protein synthesis indicate that decreased proteasomal activity inhibits protein synthesis as a feedback inhibition loop. <i>Lower proteasomal activity inhibits protein turnover rates (proteostasis-imbalance) by inhibiting both protein- synthesis and degradation</i>.</p

    CFTR ion channel activity modulators cannot restore SHS impaired bacterial phagocytosis and limit survival.

    No full text
    <p>(A) RAW264.7 cells were seeded on a 24-well plate and treated overnight with CFTR(inh)-172 (10 μM). In addition, cells were infected with <i>PA01</i>-GFP (MOI 10) for 150min followed by microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 6, white bar = 20μm). (B) Next we quantified (from 2A) the percentage of macrophages that were infected with <i>PA01</i>-GFP and found that inhibiting CFTR ion channel activity does not impair bacterial phagocytosis. (C) RAW264.7 cells were seeded on a 24-well plate and treated overnight with the flavonoids, Rutin Hydrate (RH, 10μM) or Quercetin (Q, 10μM). Cells were also infected with <i>PA01</i>-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150 min followed by microscopy. The representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 6, white bar = 20μm). (D) The quantification (from 2C) of bacterial phagocytosis shows significant (*p<0.05, **p<0.01) increase in Q or RH and CSE treated groups as compared to the CSE treated control group. But this change is not sufficient to restore SHS impaired bacterial phagocytosis to the levels seen in the control group. (E) In a separate experiment (similar to one described in 2C), media (100μl) was collected and spread on 2% LB agar plates. The plates were incubated overnight at 37°C and colony forming unit (CFU) counts were used to quantify bacterial survival. CSE treatment significantly (**p<0.01) improves bacterial survival, but RH or Q treatment of CSE group does not significantly affect bacterial counts.</p

    Salubrinal corrects proteostasis-imbalance by inhibiting the accumulation of ubiquitinated proteins.

    No full text
    <p>(<b>A</b>) The HBE cells were treated with MG-132 (1 µM) for 2 hours followed by treatment with (salubrinal, 50 µM) for another 22 hours and accumulation of ubiquitinated proteins was detected by Ub-immunostaining. The bottom panel shows the spearman's correlation coefficient analysis of fluorescence intensity. Data (n = 4) indicate a significant reduction in MG-132 induced ubiquitinated-protein accumulation by salubrinal, indicating its potential therapeutic use in treating age-related proteostasis-imbalance. (<b>B</b>) The HBE cells were treated with MG-132 and/or salubrinal (as in A) and the total protein lysates were immunoblotted for Ub, NFκB and β-actin. Data shows that salubrinal controls ubiquitin accumulation and NFκB induction. <i>Salubrinal may be effective in controlling age-related proteostasis-imbalance and inflammatory response</i>.</p

    Secondhand cigarette smoke exposure impairs bacterial phagocytosis and improves survival.

    No full text
    <p>(A) RAW264.7 cells were seeded on a 24-well plate and infected with <i>PA01</i>-GFP (multiplicity of infection, MOI 10) and/or treated with cigarette smoke extract (CSE; 10%) for 150 min as a secondhand cigarette smoke (SHS) exposure model, followed by microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (B) CSE treatment as above, significantly (**p<0.01) impairs bacterial phagocytosis in RAW264.7 cells. (C) C57BL6 mice were exposed to either room-air (control) or SHS for 5 days. Mice were infected with intra-tracheal (i.t.) <i>PA01</i>-GFP (2x10<sup>6</sup> at the indicated time points on the scale), followed by shredding of lungs for bacterial survival assay. (D) Acute-SHS exposed mice show significantly (*p<0.05) higher bacterial load (colony forming units, CFU) in the shredded lungs (low speed sonication; 1 pulse 10 sec) as compared to the room-air controls (n = 4, left: bar graph, right: scatter plot of same data). (E) The C57BL6 were exposed to either room-air (control) or SHS for 3 weeks (5 days/week) and were infected with intra-tracheal (i.t.) <i>PA01</i>-GFP (2x10<sup>6</sup> at the indicated time points on the scale). Lungs were shredded and assessed for bacterial load by quantifying colony-forming units (CFU) at the end of the 3-week period. (F) The lungs of sub-chronic- SHS exposed mice show significantly (*p<0.02, > 2 fold) greater bacterial survival (n = 4, left: bar graph, right: scatter plot of same data) as compared to the room-air controls.</p
    corecore