Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apcMin/+) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca2+ activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin–luciferase in isolated erythrocytes from apcMin/+ mice and wild-type mice (apc+/+). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apcMin/+ mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Extracellular Ca2+ removal or inhibition of Ca2+ entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca2+-entry by treatment with Ca2+-ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Following retrieval and injection into the circulation of the same mice, apcMin/+ erythrocytes were more rapidly cleared from circulating blood than apc+/+ erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apcMin/+ mice. The observations point to accelerated eryptosis and subsequent clearance of apcMin/+ erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.
Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor– and IFN regulatory factor (Irf)9–dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow–derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.