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Inhibition of inflammatory arthritis using fullerene nanomaterials.

Dellinger AL, Cunin P, Lee D, Kung AL, Brooks DB, Zhou Z, Nigrovic PA, Kepley CL - PLoS ONE (2015)

Bottom Line: It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts.Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls.In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina Greensboro, Joint School of Nanosceince and Nanoengineering, Greensboro, North Carolina, United States of America.

ABSTRACT
Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

No MeSH data available.


Related in: MedlinePlus

Mitochondrial membrane potential correlates with MC degranulation through FcγR receptors and is inhibited by fullerene derivatives.In Fig 2A, change in mitochondrial membrane potential as a function of the concentration of IC stimulus was assessed. Human MCs were stimulated with graded concentrations of preformed IgG anti-NP/NP-BSA immune complexes as indicated for 10 minutes. As a control, cells without JC-1 and cells with JC-1 plus NP IgG only (no antigen) were incubated in parallel. The above experiment is representative of two separate samples. The percent of degranulation from these cells was 23%, 32%, 39%, and 45% respectively. Mitochondrial membrane polarization was quantified by cytofluorimetry (FL2 channel) using FACs analysis as described above. As seen in Fig 2B, change in mitochondrial membrane potential as a function of time with fixed concentration of IC stimulus was assessed. Human MCs were stimulated with 8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA of preformed IgG anti-NP/NP-BSA IC for the indicated times. As a control, cells without JC-1 were incubated in parallel. In Fig 2C, Fullerene derivatives inhibit IC-induced increases in mitochondrial membrane potential. Mast cells were incubated overnight with ALM or TGA (10 μg/ml) or media only. The next day cells were challenged with media containing JC-1 probe for 10 minutes at 37°C with or without IC (as in A). After 10 minutes cells were washed with cold PBS, centrifuged and the JC-1 aggregates detected using the FL2. The above experiment is representative of three separate samples. As shown in Fig 2D fullerene derivatives inhibit IC-induced elevations in intracellular ROS levels. Mast cells were incubated overnight with fullerene derivatives, washed and DCF-DA added to cells for 30 minutes at 37°C. After washing cells were activated with optimal concentrations of IC and the fluorescence intensity measured at 525nm after establishing baseline. Figs. show representative numbers from duplicate samples for each condition and are representative of three separate MC cultures. Fig 2E shows that fullerene derivatives can block Fcγ receptor mediated activation of the MC transcription factor NF-κB. Mast cells were incubated with or without fullerene derivatives (10 μg/ml) overnight, washed, and challenged with IC for 24 hours. After washing, in-cell Westerns were performed using the manufacturers protocol. Control wells (those without primary antibodies) were reserved as a source for background well intensity. Further controls were cells incubated without fullerene derivatives or IC. Results represent results from two separate experiments.
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pone.0126290.g002: Mitochondrial membrane potential correlates with MC degranulation through FcγR receptors and is inhibited by fullerene derivatives.In Fig 2A, change in mitochondrial membrane potential as a function of the concentration of IC stimulus was assessed. Human MCs were stimulated with graded concentrations of preformed IgG anti-NP/NP-BSA immune complexes as indicated for 10 minutes. As a control, cells without JC-1 and cells with JC-1 plus NP IgG only (no antigen) were incubated in parallel. The above experiment is representative of two separate samples. The percent of degranulation from these cells was 23%, 32%, 39%, and 45% respectively. Mitochondrial membrane polarization was quantified by cytofluorimetry (FL2 channel) using FACs analysis as described above. As seen in Fig 2B, change in mitochondrial membrane potential as a function of time with fixed concentration of IC stimulus was assessed. Human MCs were stimulated with 8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA of preformed IgG anti-NP/NP-BSA IC for the indicated times. As a control, cells without JC-1 were incubated in parallel. In Fig 2C, Fullerene derivatives inhibit IC-induced increases in mitochondrial membrane potential. Mast cells were incubated overnight with ALM or TGA (10 μg/ml) or media only. The next day cells were challenged with media containing JC-1 probe for 10 minutes at 37°C with or without IC (as in A). After 10 minutes cells were washed with cold PBS, centrifuged and the JC-1 aggregates detected using the FL2. The above experiment is representative of three separate samples. As shown in Fig 2D fullerene derivatives inhibit IC-induced elevations in intracellular ROS levels. Mast cells were incubated overnight with fullerene derivatives, washed and DCF-DA added to cells for 30 minutes at 37°C. After washing cells were activated with optimal concentrations of IC and the fluorescence intensity measured at 525nm after establishing baseline. Figs. show representative numbers from duplicate samples for each condition and are representative of three separate MC cultures. Fig 2E shows that fullerene derivatives can block Fcγ receptor mediated activation of the MC transcription factor NF-κB. Mast cells were incubated with or without fullerene derivatives (10 μg/ml) overnight, washed, and challenged with IC for 24 hours. After washing, in-cell Westerns were performed using the manufacturers protocol. Control wells (those without primary antibodies) were reserved as a source for background well intensity. Further controls were cells incubated without fullerene derivatives or IC. Results represent results from two separate experiments.

Mentions: Unstable mitochondrial membrane potential regulates ROS production [47]. Our previous work strongly suggested that fullerene derivatives inhibited degranulation through a pathway involving mitochondrial signaling proteins [24] and ALM is specifically designed to target mitochondrial membranes [27]. However, no studies have examined the role of mitochondrial membrane potential or fullerene derivatives in IC mediator release from human MC. Given that increases in MC mitochondrial membrane potential closely paralleled degranulation and previous studies suggested mitochondrial signaling pathways were affected by fullerene derivatives, it was hypothesized that the inhibitory effect of fullerene derivatives on MC degranulation may involve modulation of the mitochondrial membrane potential response. Initial studies demonstrated that MC mitochondrial membrane potential was dependent on dose (Fig 2A) and time (Fig 2B) of the degranulation stimulus using IC. As seen in Fig 2C, MC incubated with fullerene derivatives prior to challenge with optimal concentrations of IC demonstrated a decrease in mitochondrial membrane potential compared to untreated MC. ALM and TGA also inhibited IC-induced increases in ROS activity (Fig 2D). Lastly, NF-κB, which regulates genes controlling the amount of ROS and TNF-α in the cell [48,49], was down-regulated in IC-treated MC pre-incubated with fullerene derivatives (Fig 2E). Thus, decreased MC cellular activation through IC is due in part to decreased mitochondrial membrane potential, ROS production, and NF-κB activation. Two of the overall best inhibitors of these parameters included ALM and TGA, which were chosen for further study.


Inhibition of inflammatory arthritis using fullerene nanomaterials.

Dellinger AL, Cunin P, Lee D, Kung AL, Brooks DB, Zhou Z, Nigrovic PA, Kepley CL - PLoS ONE (2015)

Mitochondrial membrane potential correlates with MC degranulation through FcγR receptors and is inhibited by fullerene derivatives.In Fig 2A, change in mitochondrial membrane potential as a function of the concentration of IC stimulus was assessed. Human MCs were stimulated with graded concentrations of preformed IgG anti-NP/NP-BSA immune complexes as indicated for 10 minutes. As a control, cells without JC-1 and cells with JC-1 plus NP IgG only (no antigen) were incubated in parallel. The above experiment is representative of two separate samples. The percent of degranulation from these cells was 23%, 32%, 39%, and 45% respectively. Mitochondrial membrane polarization was quantified by cytofluorimetry (FL2 channel) using FACs analysis as described above. As seen in Fig 2B, change in mitochondrial membrane potential as a function of time with fixed concentration of IC stimulus was assessed. Human MCs were stimulated with 8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA of preformed IgG anti-NP/NP-BSA IC for the indicated times. As a control, cells without JC-1 were incubated in parallel. In Fig 2C, Fullerene derivatives inhibit IC-induced increases in mitochondrial membrane potential. Mast cells were incubated overnight with ALM or TGA (10 μg/ml) or media only. The next day cells were challenged with media containing JC-1 probe for 10 minutes at 37°C with or without IC (as in A). After 10 minutes cells were washed with cold PBS, centrifuged and the JC-1 aggregates detected using the FL2. The above experiment is representative of three separate samples. As shown in Fig 2D fullerene derivatives inhibit IC-induced elevations in intracellular ROS levels. Mast cells were incubated overnight with fullerene derivatives, washed and DCF-DA added to cells for 30 minutes at 37°C. After washing cells were activated with optimal concentrations of IC and the fluorescence intensity measured at 525nm after establishing baseline. Figs. show representative numbers from duplicate samples for each condition and are representative of three separate MC cultures. Fig 2E shows that fullerene derivatives can block Fcγ receptor mediated activation of the MC transcription factor NF-κB. Mast cells were incubated with or without fullerene derivatives (10 μg/ml) overnight, washed, and challenged with IC for 24 hours. After washing, in-cell Westerns were performed using the manufacturers protocol. Control wells (those without primary antibodies) were reserved as a source for background well intensity. Further controls were cells incubated without fullerene derivatives or IC. Results represent results from two separate experiments.
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Related In: Results  -  Collection

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pone.0126290.g002: Mitochondrial membrane potential correlates with MC degranulation through FcγR receptors and is inhibited by fullerene derivatives.In Fig 2A, change in mitochondrial membrane potential as a function of the concentration of IC stimulus was assessed. Human MCs were stimulated with graded concentrations of preformed IgG anti-NP/NP-BSA immune complexes as indicated for 10 minutes. As a control, cells without JC-1 and cells with JC-1 plus NP IgG only (no antigen) were incubated in parallel. The above experiment is representative of two separate samples. The percent of degranulation from these cells was 23%, 32%, 39%, and 45% respectively. Mitochondrial membrane polarization was quantified by cytofluorimetry (FL2 channel) using FACs analysis as described above. As seen in Fig 2B, change in mitochondrial membrane potential as a function of time with fixed concentration of IC stimulus was assessed. Human MCs were stimulated with 8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA of preformed IgG anti-NP/NP-BSA IC for the indicated times. As a control, cells without JC-1 were incubated in parallel. In Fig 2C, Fullerene derivatives inhibit IC-induced increases in mitochondrial membrane potential. Mast cells were incubated overnight with ALM or TGA (10 μg/ml) or media only. The next day cells were challenged with media containing JC-1 probe for 10 minutes at 37°C with or without IC (as in A). After 10 minutes cells were washed with cold PBS, centrifuged and the JC-1 aggregates detected using the FL2. The above experiment is representative of three separate samples. As shown in Fig 2D fullerene derivatives inhibit IC-induced elevations in intracellular ROS levels. Mast cells were incubated overnight with fullerene derivatives, washed and DCF-DA added to cells for 30 minutes at 37°C. After washing cells were activated with optimal concentrations of IC and the fluorescence intensity measured at 525nm after establishing baseline. Figs. show representative numbers from duplicate samples for each condition and are representative of three separate MC cultures. Fig 2E shows that fullerene derivatives can block Fcγ receptor mediated activation of the MC transcription factor NF-κB. Mast cells were incubated with or without fullerene derivatives (10 μg/ml) overnight, washed, and challenged with IC for 24 hours. After washing, in-cell Westerns were performed using the manufacturers protocol. Control wells (those without primary antibodies) were reserved as a source for background well intensity. Further controls were cells incubated without fullerene derivatives or IC. Results represent results from two separate experiments.
Mentions: Unstable mitochondrial membrane potential regulates ROS production [47]. Our previous work strongly suggested that fullerene derivatives inhibited degranulation through a pathway involving mitochondrial signaling proteins [24] and ALM is specifically designed to target mitochondrial membranes [27]. However, no studies have examined the role of mitochondrial membrane potential or fullerene derivatives in IC mediator release from human MC. Given that increases in MC mitochondrial membrane potential closely paralleled degranulation and previous studies suggested mitochondrial signaling pathways were affected by fullerene derivatives, it was hypothesized that the inhibitory effect of fullerene derivatives on MC degranulation may involve modulation of the mitochondrial membrane potential response. Initial studies demonstrated that MC mitochondrial membrane potential was dependent on dose (Fig 2A) and time (Fig 2B) of the degranulation stimulus using IC. As seen in Fig 2C, MC incubated with fullerene derivatives prior to challenge with optimal concentrations of IC demonstrated a decrease in mitochondrial membrane potential compared to untreated MC. ALM and TGA also inhibited IC-induced increases in ROS activity (Fig 2D). Lastly, NF-κB, which regulates genes controlling the amount of ROS and TNF-α in the cell [48,49], was down-regulated in IC-treated MC pre-incubated with fullerene derivatives (Fig 2E). Thus, decreased MC cellular activation through IC is due in part to decreased mitochondrial membrane potential, ROS production, and NF-κB activation. Two of the overall best inhibitors of these parameters included ALM and TGA, which were chosen for further study.

Bottom Line: It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts.Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls.In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina Greensboro, Joint School of Nanosceince and Nanoengineering, Greensboro, North Carolina, United States of America.

ABSTRACT
Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

No MeSH data available.


Related in: MedlinePlus