The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-␣ expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-␣ expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMPactivated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/ STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.Resident tissue macrophages exhibit a tremendous plasticity in their response to external stimuli. In particular, the local metabolic and immune microenvironment contributes to this plasticity (1). In response to different stimuli, macrophages express distinct patterns of surface receptors and metabolic enzymes that ultimately generate the diversity of macrophage functions. Although the precise characterization of macrophage diversity is continuing to evolve, two distinct polarization states of macrophages, M1 and M2, have been characterized (1, 2). LPS and IFN␥ can promote macrophage differentiation to a "classical" or M1 phenotype (1). The M1 activation pattern is associated with tissue destruction and inflammation and is responsible for up-regulating pro-inflammatory cytokines and increasing production of reactive nitrogen species and reactive oxygen species (1). In contrast, the "alternative" or M2 activation phenotype of macrophages is induced in response to IL-4 and IL-13. M2-polarized macrophages dampen the inflammatory process by producing anti-inflammatory factors, such as IL-10 and TGF␤. M2 macrophages also up-regulate mannose receptors, such as the macrophage mannose receptor (MMR) 2 and mannose receptor, C type 2 (Mrc2c), IL-1 receptor antagonist (I...

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mRNAs encoding proinflammatory chemokines are regulated posttranscriptionally via adenine-uridine-rich sequences (AREs) located in the 3′ untranslated region of the message, which are recognized by sequence-specific RNA-binding proteins. One ARE binding protein, tristetraprolin (TTP), has been implicated in regulating the stability of several ARE-containing mRNAs, including those encoding TNF-α and GM-CSF. In the present report we examined the role of TTP in regulating the decay of the mouse chemokine KC (CXCL1) mRNA. Using tetR-regulated control of transcription in TTP-deficient HEK293 cells, KC mRNA half-life was markedly decreased in the presence of TTP. Deletion and site-specific mutagenesis were used to identify multiple AUUUA sequence determinants responsible for TTP sensitivity. Although a number of studies suggest that the destabilizing activity of TTP is subject to modulation in response to ligands of Toll/IL-1 family receptors, decay mediated by TTP in 293 cells was not sensitive to stimulation with IL-1α. Using primary macrophages from wild-type and TTP-deficient mice, KC mRNA instability was found to be highly dependent on TTP. Furthermore, LPS-mediated stabilization of KC mRNA is blocked by inhibition of the p38 MAPK in macrophages from wild-type but not TTP-deficient mice. These findings demonstrate that TTP is the predominant regulator of KC mRNA decay in mononuclear phagocytes acting via multiple 3′-untranslated region-localized AREs. Nevertheless, KC mRNA remains highly unstable in cells that do not express TTP, suggesting that additional determinants of instability and stimulus sensitivity may operate in cell populations where TTP is not expressed.

Background Correlative evidence indicates that apoptosis is associated with the progression of alcoholic liver disease. If apoptosis contributes to ethanol-induced steatohepatitis and/or fibrosis, then mice deficient in Bid, a key pro-apoptotic Bcl-2 family member, or mice treated with a pan-caspase inhibitor (VX166) should be resistant to ethanol-induced liver injury. Methods This hypothesis was tested in mice using a model of chronic, heavy ethanol-induced liver injury, as well as in a model in which moderate ethanol feeding accelerated the appearance of early markers of hepatic fibrosis in response to acute carbon tetrachloride exposure. Results Chronic ethanol feeding to mice increased TUNEL- and cytokeratin 18-positive cells in the liver, as well as the expression of receptor interacting protein kinase 3 (RIP3), a marker of necroptosis. In this model, Bid−/− mice or wild-type mice treated with VX166 were protected from ethanol-induced apoptosis, but not ethanol-induced RIP3 expression. Bid-deficiency or inhibition of caspase activity did not protect mice from ethanol-induced increases in plasma alanine and aspartate amino transferase activity, steatosis or mRNA expression of some inflammatory cytokines. Moderate ethanol feeding to mice enhanced the response of mice to acute carbon tetrachloride exposure, resulting in increased expression of α-smooth muscle actin and accumulation of extracellular matrix protein. VX166-treatment attenuated ethanol- mediated acceleration of these early indicators of carbon tetrachloride-induced hepatic fibrosis, decreasing the expression of α-smooth muscle actin and the accumulation of extracellular matrix protein. Conclusion Ethanol-induced apoptosis of hepatocytes was mediated by Bid. Apoptosis played a critical role in the accelerating the appearance of early markers of carbon tetrachloride-induced fibrosis by moderate ethanol, but did not contribute to ethanol-induced hepatocyte injury, steatosis or expression of mRNA for some inflammatory cytokines.

We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.