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Consistent inhibition of cyclooxygenase drives macrophages towards the inflammatory phenotype.

Na YR, Yoon YN, Son D, Jung D, Gu GJ, Seok SH - PLoS ONE (2015)

Bottom Line: Thus alterations of macrophage function can have unexpected pathological results.Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated.YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype.

View Article: PubMed Central - PubMed

Affiliation: Macrophage Lab, Department of Microbiology and Immunology, and Institute of Endemic Disease, Seoul National University College of Medicine, 103 Daehak-ro, Chongno-gu, Seoul 110-799, South Korea.

ABSTRACT
Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.

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Cytokine responses in COX inhibited bone-marrow-derived macrophages.Panel A: Time line of macrophage differentiation with COX inhibition and LPS stimulation. COX inhibitors were treated in two modes: SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) were treated at 30 min before LPS stimulation (acute) or during whole differentiation period (chronic). Panels B-G: Macrophage TNFα (Panels B, C), IL-12p70 (Panels D, E) and IL-10 (Panels F, G) productions analyzed by ELISA. COX inhibitors were treated chronically (Panels B, D, F) or acutely (Panels C, E, G). LPS was treated at 100 ng/ml and culture medium was collected after 4 h (TNFα) and 24 h (IL-12p70 and IL-10) incubation for analysis. Results represent means ± SE of three independent experiments. Statistical analysis was performed by one-way ANOVA. *P<0.05, ***P<0.001.
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pone.0118203.g001: Cytokine responses in COX inhibited bone-marrow-derived macrophages.Panel A: Time line of macrophage differentiation with COX inhibition and LPS stimulation. COX inhibitors were treated in two modes: SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) were treated at 30 min before LPS stimulation (acute) or during whole differentiation period (chronic). Panels B-G: Macrophage TNFα (Panels B, C), IL-12p70 (Panels D, E) and IL-10 (Panels F, G) productions analyzed by ELISA. COX inhibitors were treated chronically (Panels B, D, F) or acutely (Panels C, E, G). LPS was treated at 100 ng/ml and culture medium was collected after 4 h (TNFα) and 24 h (IL-12p70 and IL-10) incubation for analysis. Results represent means ± SE of three independent experiments. Statistical analysis was performed by one-way ANOVA. *P<0.05, ***P<0.001.

Mentions: To investigate the effects of COX inhibition on macrophages, cultured bone marrow cells were exposed to various COX inhibitors, including the COX-1–specific inhibitor SC-560 (0.5 μM), COX-2–specific inhibitor NS-398 (50 μM) and COX-1/2 inhibitor indomethacin (100 μM). Timeline shows two modes of drug treatment (Fig. 1A); chronic exposure was started from bone marrow cell seeding and continued during 7 days of macrophage differentiation with fresh drug treatment at day 3. Acute exposure was started at 30 min before LPS treatment into the mature macrophages. When macrophages were stimulated with LPS, they produced significantly more TNFα in chronic NS-398 and indomethacin treated groups (Fig. 1B). Short-term 30 min pre-treatment of COX inhibitors before LPS stimulation reduced TNFα in both groups (Fig. 1C). In case of IL-12p70, SC-560 and indomethacin treated macrophages produced more amounts than control only when were exposed during whole 7 days of differentiation (Fig. 1D, E). Inversely, IL-10 secretion was lower in chronic NS-398 and indomethacin-treated macrophages (Fig. 1F). To enlarge basal state cytokine productions of macrophages, we investigated intracellular cytokine accumulation in F4/80+CD11b+ mature macrophages during 4 hours with Brefeldin A (S1 Fig.). FACS data clearly showed that chronic COX inhibitor treated macrophages synthesized more TNFα but less IL-10 compared with control in a basal state. Next, to confirm whether TLR early signaling pathway is relevant with TNFα production, we examined phospho-IκBα and phospho-p65 from whole cell lysates after LPS stimulation using Western blotting (Fig. 2A). Accordingly, both chronic COX-2 as well as COX-1/2 inhibited macrophages exhibited reduced total IκBα, as well as enhanced p65 phosphoylation upon LPS stimulation compared with the control. Enhanced NF-κB activity directly connected to TNFα production ability because enhanced TNFα productions were recovered when NF-kB inhibitor was treated before LPS stimulation in NS-398 and indomethacin groups (Fig. 2B). Collectively, these results indicated that macrophages lacking COXs activities during differentiation are more immune activated.


Consistent inhibition of cyclooxygenase drives macrophages towards the inflammatory phenotype.

Na YR, Yoon YN, Son D, Jung D, Gu GJ, Seok SH - PLoS ONE (2015)

Cytokine responses in COX inhibited bone-marrow-derived macrophages.Panel A: Time line of macrophage differentiation with COX inhibition and LPS stimulation. COX inhibitors were treated in two modes: SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) were treated at 30 min before LPS stimulation (acute) or during whole differentiation period (chronic). Panels B-G: Macrophage TNFα (Panels B, C), IL-12p70 (Panels D, E) and IL-10 (Panels F, G) productions analyzed by ELISA. COX inhibitors were treated chronically (Panels B, D, F) or acutely (Panels C, E, G). LPS was treated at 100 ng/ml and culture medium was collected after 4 h (TNFα) and 24 h (IL-12p70 and IL-10) incubation for analysis. Results represent means ± SE of three independent experiments. Statistical analysis was performed by one-way ANOVA. *P<0.05, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4334507&req=5

pone.0118203.g001: Cytokine responses in COX inhibited bone-marrow-derived macrophages.Panel A: Time line of macrophage differentiation with COX inhibition and LPS stimulation. COX inhibitors were treated in two modes: SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) were treated at 30 min before LPS stimulation (acute) or during whole differentiation period (chronic). Panels B-G: Macrophage TNFα (Panels B, C), IL-12p70 (Panels D, E) and IL-10 (Panels F, G) productions analyzed by ELISA. COX inhibitors were treated chronically (Panels B, D, F) or acutely (Panels C, E, G). LPS was treated at 100 ng/ml and culture medium was collected after 4 h (TNFα) and 24 h (IL-12p70 and IL-10) incubation for analysis. Results represent means ± SE of three independent experiments. Statistical analysis was performed by one-way ANOVA. *P<0.05, ***P<0.001.
Mentions: To investigate the effects of COX inhibition on macrophages, cultured bone marrow cells were exposed to various COX inhibitors, including the COX-1–specific inhibitor SC-560 (0.5 μM), COX-2–specific inhibitor NS-398 (50 μM) and COX-1/2 inhibitor indomethacin (100 μM). Timeline shows two modes of drug treatment (Fig. 1A); chronic exposure was started from bone marrow cell seeding and continued during 7 days of macrophage differentiation with fresh drug treatment at day 3. Acute exposure was started at 30 min before LPS treatment into the mature macrophages. When macrophages were stimulated with LPS, they produced significantly more TNFα in chronic NS-398 and indomethacin treated groups (Fig. 1B). Short-term 30 min pre-treatment of COX inhibitors before LPS stimulation reduced TNFα in both groups (Fig. 1C). In case of IL-12p70, SC-560 and indomethacin treated macrophages produced more amounts than control only when were exposed during whole 7 days of differentiation (Fig. 1D, E). Inversely, IL-10 secretion was lower in chronic NS-398 and indomethacin-treated macrophages (Fig. 1F). To enlarge basal state cytokine productions of macrophages, we investigated intracellular cytokine accumulation in F4/80+CD11b+ mature macrophages during 4 hours with Brefeldin A (S1 Fig.). FACS data clearly showed that chronic COX inhibitor treated macrophages synthesized more TNFα but less IL-10 compared with control in a basal state. Next, to confirm whether TLR early signaling pathway is relevant with TNFα production, we examined phospho-IκBα and phospho-p65 from whole cell lysates after LPS stimulation using Western blotting (Fig. 2A). Accordingly, both chronic COX-2 as well as COX-1/2 inhibited macrophages exhibited reduced total IκBα, as well as enhanced p65 phosphoylation upon LPS stimulation compared with the control. Enhanced NF-κB activity directly connected to TNFα production ability because enhanced TNFα productions were recovered when NF-kB inhibitor was treated before LPS stimulation in NS-398 and indomethacin groups (Fig. 2B). Collectively, these results indicated that macrophages lacking COXs activities during differentiation are more immune activated.

Bottom Line: Thus alterations of macrophage function can have unexpected pathological results.Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated.YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype.

View Article: PubMed Central - PubMed

Affiliation: Macrophage Lab, Department of Microbiology and Immunology, and Institute of Endemic Disease, Seoul National University College of Medicine, 103 Daehak-ro, Chongno-gu, Seoul 110-799, South Korea.

ABSTRACT
Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.

Show MeSH
Related in: MedlinePlus