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IL-1β and TNF-α are important proinflammatory cytokines that respond to mutated self-antigens of tissue damage and exogenous pathogens. The endoplasmic reticulum (ER) stress and unfolded protein responses are related to the induction of proinflammatory cytokines. However, the detailed molecular pathways by which ER stress mediates cytokine gene expression have not been investigated. In this study, we found that ER stress–induced inositol-requiring enzyme (IRE)1α activation differentially regulates proinflammatory cytokine gene expression via activation of glycogen synthase kinase (GSK)-3β and X-box binding protein (XBP)-1. Surprisingly, IL-1β gene expression was modulated by IRE1α-mediated GSK-3β activation, but not by XBP-1. However, IRE1α-mediated XBP-1 splicing regulated TNF-α gene expression. SB216763, a GSK-3 inhibitor, selectively inhibited IL-1β gene expression, whereas the IRE1α RNase inhibitor STF083010 suppressed only TNF-α production. Additionally, inhibition of GSK-3β greatly increased IRE1α-dependent XBP-1 splicing. Our results identify an unsuspected differential role of downstream mediators GSK-3β and XBP-1 in ER stress–induced IRE1α activation that regulates cytokine production through signaling cross-talk. These results have important implications in the regulation of inflammatory pathways during ER stress, and they suggest novel therapeutic targets for diseases in which meta-inflammation plays a key role.

The mechanisms underlying pathophysiological states such as metabolic syndrome and obesity include endoplasmic reticulum (ER) stress and aberrant inflammatory responses. ER stress results from the accumulation of misfolded proteins during stress conditions. However, the precise mechanisms by which ER stress modulates inflammation remain incompletely understood. In this study, we hypothesized that ER stress alone could represent a sufficient signal for the modulation of inflammasome-dependent cytokine responses. We found that several ER stress-inducing chemicals and the free fatty acid palmitate can trigger IL-1β secretion in various cell types, including monocytic leukemia cells, primary macrophages and differentiated adipocytes. We show that ER stress primes cells for the expression of pro-IL-1β via NF-κB activation and promotes IL-1β secretion. Enhanced IL-1β secretion depended on the activation of the NLRP3 inflammasome through a mechanism involving reactive oxygen species formation and activation of thioredoxin-interacting protein. Chemical chaperone treatment and the pharmacological application of carbon monoxide inhibited IL-1β secretion in response to ER stress. Our results provide a mechanistic link between ER stress and the regulation of inflammation, and suggest that modulation of ER stress may provide a therapeutic opportunity to block progression of low grade chronic inflammation to metabolic syndrome.

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Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.