Chronic Toll-like receptor 4 stimulation in skin induces inflammation,macrophage activation,transforming growth factor beta signature gene expression,and fibrosis

Introduction

The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis.

Methods

The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry.

Results

We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes.

Conclusions

We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.     

PTEN Negatively Regulates MAPK Signaling during Caenorhabditis elegans Vulval Development

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP(3) lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.     

Tight-binding inhibitors efficiently inactivate both reaction centers of monomeric Plasmodium falciparum glyoxalase 1

Glucose consumption and therefore methylglyoxal production of human erythrocytes increase significantly upon infection with malaria parasites. The glyoxalase systems of the host-parasite unit cope with this metabolic challenge by catalyzing the removal of harmful methylglyoxal. Thus, glyoxalase 1 from the malaria parasite Plasmodium falciparum (PfGlo1) could be a promising drug target. However, the enzyme has two different active sites and their simultaneous inactivation is considered challenging. Here, we describe the inactivation of PfGlo1 by two glyoxalase-specific tight-binding inhibitors with nanomolar K(i)(app) values and noncompetitive inhibition patterns. The inhibitors do not discriminate between the high-affinity and the high-activity conformations of PfGlo1, but seem to stabilize or trigger a conformational change in analogy with the substrate. In summary, we have characterized the most potent inhibitors of PfGlo1 known to date.     

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

Changes in the kinetics and emission spectrum on mutation of the chromophore-binding platform in Vibrio harveyi luciferase

The recently proposed model for the bacteria luciferase-flavin mononucleotide complex identifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazine ring of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between the isopropyl side chain of alphaVal173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophore represents an important target to test the validity of the proposed model. Here, structure-function analysis of luciferase variants carrying single point mutations at position alpha173 have verified the functional layout of the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease in the stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account for changes in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position alpha173 to increase its polarity confirmed that alphaVal173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of the isoalloxazine chromophore (alphaA75G/C106V) into the alphaV173A, alphaV173C, alphaV173T, and alphaV173S mutants led to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted the emission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emission wavelength spectrum provides the basis toward engineering luciferase variants that emit different light colors with the proposed flavin-luciferase model complex as a design reference.     

Cyclooxygenase inhibitors modulate NK activities that control metastatic disease

Cyclooxygenase (COX) inhibitors have demonstrated efficacy in models of human cancer but the relevant mechanisms have not all been elucidated. Both Cox-dependent as well as Cox-independent mechanisms have been implicated. Using a syngeneic model of metastatic breast cancer, we have investigated the effect of Cox inhibitors on NK functions that are critical to the control of metastatic disease. NK recognition of target cells is governed by a balance of activating and inhibiting receptors that bind ligands including MHC class I. We now show that treatment of tumor cells with the nonselective COX-1/COX-2 inhibitor indomethacin or the selective COX-2 inhibitor celecoxib leads to decreased expression of the MHC class I molecules L^d and K^d . Downregulated class I expression is associated with concomitant increased sensitivity to NK cell-mediated lysis. Both COX inhibitors limit tumor metastasis and this therapeutic effect is dependent on NK but not T cell function. Antimetastatic activity is also lost in the absence of interferon- (IFN-). Both COX inhibitors also suppress local tumor growth of subcutaneously implanted mammary tumor cells in immune competent Balb/cByJ mice. This therapeutic activity is lost in the absence of either CD4+ or CD8+ T cells, but is not compromised by the loss of NK activity. Thus, the mechanism of tumor inhibition differs in the context of local versus metastatic disease. Taken together, these findings are consistent with a mechanism not previously described, whereby COX inhibitors may relieve MHC-mediated inhibition of NK cytotoxicity leading to recognition and lysis of metastatic tumor cells.     

Inhibition of pMAPK14 Overcomes Resistance to Sorafenib in Hepatoma Cells with Hepatitis B Virus

Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.