Immune Netw.  2013 Aug;13(4):141-147. 10.4110/in.2013.13.4.141.

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

• Kim EH
• Won JH
• Hwang I
• Yu JW

• KMID: 2150779
• DOI: http://doi.org/10.4110/in.2013.13.4.141

Abstract

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.

Keyword

Cobalt chloride (CoCl2); Hypoxia; NLRP3; Inflammasome; Caspase-1

MeSH Terms

Animals
Anoxia
Brain
Cobalt
Cytokines
Inflammation
Macrophages
Mice
Neuroglia
Cobalt
Cytokines

Figure

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  Figure 1 Hypoxia does not activate caspase-1 in BV-2 cells. Cultural supernatants or lysates were immunoblotted as indicated. (A) BV-2 cells were resuspended in glucose-free media and incubated in hypoxic chamber for the indicated times. (B) BV-2 cells were treated with OGD as described in (A) for 5 h in the presence or absence of zVAD (20µM). (C) BV-2 cells were treated with LPS (0.25µg/ml) for 3 h, followed by treatment with CoCl2 (100µM) in the presence or absence of zVAD (20 µM) for 15 h.

• 

  Figure 2 CoCl2-induced hypoxia does not induce caspase-1 activation in primary mixed glial cultures. (A) Immunoblots for the indicated antibodies in cultural supernatants or lysates of brain mixed glial cells treated with LPS (0.25µg/ml) with or without CoCl2 (100µM) for the indicated times or LPS followed with nigericin (5 mM, final 45 min) as indicated. (B) Mouse bone marrow-derived macrophages were treated with LPS (0.25µg/ml) for 3 h, followed by treatment with CoCl2 (100µM) or nigericin (5µM, 45 min) as indicated. (C) PMA-differentiated THP-1 cells were treated with CoCl2 (100µM) or LPS (0.5µ g/ml, 6 h). Soluble lysates and supernatants were immunoblotted as indicated antibodies. Note that the processed caspase-1 p10 or p20 band is detected depending on the p10- or p20-specific anti-caspase-1 antibody.

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  Figure 3 CoCl2-induced hypoxia attenuates NLRP3-dependent caspase-1 activation in primary mixed glial cultures. (A, B) Immunoblots for the indicated antibodies in cultural supernatants or lysates of brain mixed glial cells treated with LPS (0.25µg/ml) with or without CoCl2 (100µM) for 7 h, followed by treatment with ATP (5 mM, 45 min) or nigericin (5µM, 45 min), or transfected with poly dA:dT (1µg, 6 h) with or without CoCl2 (100µM). (C) Immunoblots for the indicated antibodies in cultural supernatants or lysates of bone marrow-derived macrophages treated with LPS (0.25µg/ml) with or without CoCl2 (100µM) for 7 h, followed by treatment with ATP (5 mM, 45 min) or nigericin (5µM, 45 min).

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  Figure 4 Time course effect of CoCl2 on the NLRP3 inflammasome activity. (A) Immunoblots for the indicated antibodies in cultural supernatants or lysates of brain mixed glial cells treated with LPS (0.25 µg/ml, 7 h) with or without CoCl2 (100µM, indicated times), followed by treatment with ATP (5 mM, 45 min). Note that the CoCl2 of the sixth lane was cotreated with ATP. (B) Immunoblots for the indicated antibodies in cultural supernatants or lysates of brain mixed glial cells treated with LPS (0.25µ g/ml) with or without CoCl2 (100µM) for the indicated times, followed by treatment with ATP (5 mM, 45 min).

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  Figure 5 CoCl2-induced hypoxia reduces NLRP3-dependent caspase-1 activation in primary microglial cells. (A) Flow cytometric analysis of mouse brain mixed glial cells (upper) and isolated microglial cells (lower) using anti-CD11b-PE and anti-F4/80 antigen-APC antibody. (B) Immunoblots for the indicated antibodies in cultural supernatants of brain mixed glial cells or isolated microglial cells treated with LPS (0.25µg/ml) with or without CoCl2 (100µM) for 7 h, followed by treatment with ATP (5 mM, 45 min).

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  Figure 6 Mitochondrial ROS is not critical for NLRP3 inflammasome activation in primary mixed glial cells. (A) Flow cytometric analysis of mouse bone marrow-derived macrophages treated with rotenone (10µM, 1 h) or ATP (3mM, 30 min) and stained with MitoSox. (B) Flow cytometric analysis of brain mixed glial cells treated with rotenone (10µM, 2 h) or LPS (0.25µg/ml) with or without CoCl2 (100 µM) for the indicated times, followed treatment with ATP (5 mM, 40 min) and stained with MitoSox.

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