Fig 1.
Copper-infused fabric dampen inflammatory gene expression.
(a) copper assay showing the concentration of copper in undiluted leachate (100%), a 1:1 ratio of leachate to normal growth media (50%) or 1:3 ratio of leachate to normal growth media (25%). (b, c) THP-1 human monocytic cells were differentiated into macrophages following incubation with 100 nM PMA overnight. Cells were then cultured with 100% copper-infused fabric (Copper 2P) or non-copper fabric (control) leachates that were collected from pre-incubating the Copper 2P and control fabrics in media for 24 hours. Next, cells were stimulated with 100 ng/ml LPS for 1, 3, and 6 hours. Non-induced cells (ϕ) were only incubated with copper 2P and control leachates, and included as a negative control. (b) IL-1β and CxCL1 production were measured in supernatants of cultured cells by ELISA. (c) Gene expression of TNF, IFN-β, IL-1β and IL-10 was evaluated by real-time PCR (Q-PCR). GAPDH was used as a reference gene to normalize the data. Data from all experiments are representative of at least 3 independent experiments. * p<0.05; ** p<0.01; **** p<0.0001.
Fig 2.
Dose-dependent effects of copper-infused fabric on inflammatory gene expression.
PMA differentiated THP-1 cells were cultures in 100% leachate, 50% leachate or 25% leachate for 24 hours, as in Fig 1a, and then stimulated with 100 ng/ml LPS for the indicated times. Non-induced cells (ϕ) have been included as a negative control. Gene expression of (a) TNF-α, (b) IFN-β and (c) IL-1β was evaluated by real-time PCR (Q-PCR). GAPDH was used as a reference gene to normalize the data and plotted as a time course for each leachate concentration (left panels) or at the peak response (3h) to show bar graphs with all three concentrations (right panel). Data from all experiments are representative of at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001.
Fig 3.
LPS driven inflammation can be attenuated following incubation with copper-infused fabric.
PMA differentiated THP-1 cells were stimulated with 100 ng/ml LPS in normal growth media for 15 min before media replacement with leachates obtained from control or copper containing fabric. 100 ng/ml LPS was added to the leachates to maintain inflammatory cytokine stimulation. Cells were then incubated for an additional 15 min (15’), 45 min (45’) or 2 hours (2h). TNF-α, IL-1β, IL-6 and IL-10 gene expression was evaluated by real-time PCR (Q-PCR). GAPDH was used as a reference gene to normalize the data. Data from all experiments are representative of at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001.
Fig 4.
Copper-infused fabrics reduce LPS induced NF-κB and IRF3 activation.
(a) PMA differentiated THP-1 cells were cultured in growth media that was pre-incubated with non-copper containing fabric (control fabric) or copper-infused fabric (Copper 2P) for 24 hours, then stimulated with 100 ng/ml LPS for the indicated times. Non-induced cells (ϕ) have been included as a negative control. Whole cell extracts were then prepared from these cells and immunoblotted for phosphor-specific IκB-α (p-IκB-α) or total IκB-α (IκB-α), and as a loading control, β-Actin. (b, c) The reporter mouse macrophage cell line RAW-Dual™ cells were cultured in growth media that was pre-incubated with non-copper containing fabric (control fabric) or copper-infused fabrics (Copper 2P) for 24 hours, then stimulated with 100 ng/ml LPS for the 18 or 24 hours. Non-induced cells (ϕ) have been included as a negative control. (b) NF-κB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm, and (c) IRF3 activation was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™, a Lucia luciferase detection reagent. Data from all experiments are representative of at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001.
Fig 5.
Copper-infused fabric reduce inflammatory signaling and proteins in LPS-stimulated macrophages.
(a) Gating strategy for intracellular flow cytometry analysis performed on PMA differentiated THP-1 cells for measurements of phospho-P65 (p-P65), phospho-S6 (p-S6), and Interleukin-1 beta (IL-1β). (b-d) PMA differentiated THP-1 cells were cultured in growth media that was pre-incubated with non-copper containing fabric (control fabric) or copper-infused fabric (Copper) and stimulated with 100 ng/ml LPS for 30 or 60 min as indicated (30’ and 60’, respectively). Non-induced cells (ϕ) have been included as a negative control. Intracellular protein expression of (b) phospho-P65 (p-P65), (c) phospho-S6 (p-S6), and (d) Interleukin-1 beta (IL-1β) were measured by flow cytometry. The panels to the right are bar graphs plotting the dMFI, which is the mean fluorescence intensity (MFI) of each sample minus that of the fluorescence minus one (FMO) control. Data from all experiments are representative of at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001.
Fig 6.
Copper rescues insulin sensitivity in L6 skeletal muscle cells.
(a) Representative images of the biosensor-transfected L6 cells upon treatment with conditioned media obtained from LPS-stimulated THP-1 derived macrophages that were incubated leachate from non-copper containing fabric (control) or copper-infused fabric (Copper) for 20 min in the presence of 100 nM insulin. (b) Percentage of change in fluorescent signal observed in biosensor- transfected cells in control and copper treated media, as in (a), in the presence or absence of 100 nM insulin. The fluorescent intensity was measured manually for the duration of 20 min. (c) Representative Western blot images and quantification of phospho-specific Akt (pAkt s473) over total Akt (tAkt) after incubation in conditioned media as in (a) for 24 hours. Data from all experiments are representative of at least 3 independent experiments. **P < 0.01.