Inflammatory Toll-like Receptor Research Articles

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Effect of LRRK2 protein and activity on stimulated cytokines in human monocytes and macrophages

Leucine-rich-repeat kinase 2 (LRRK2), a potential therapeutic target for the treatment of Parkinson’s disease (PD), is highly expressed in monocytes and macrophages and may play a role in the regulation of inflammatory pathways. To determine how LRRK2 protein levels and/or its activity modulate inflammatory cytokine/chemokine levels in human immune cells, isogenic human induced pluripotent stem cells (iPSC) with the LRRK2-activating G2019S mutation, wild-type LRRK2, and iPSC deficient in LRRK2 were differentiated to monocytes and macrophages and stimulated with inflammatory toll-like receptor (TLR) agonists in the presence and absence of LRRK2 kinase inhibitors. The effect of LRRK2 inhibitors and the effect of increasing LRRK2 levels with interferon gamma on TLR-stimulated cytokines were also assessed in primary peripheral blood-derived monocytes. Monocytes and macrophages with the LRRK2 G2019S mutation had significantly higher levels of cytokines and chemokines in tissue culture media following stimulation with TLR agonists compared to isogenic controls. Knockout of LRRK2 impaired phagocytosis but did not significantly affect TLR-mediated cytokine levels. Interferon gamma significantly increased the levels of LRRK2 and phosphorylation of its downstream Rab10 substrate, and potentiated TLR-mediated cytokine levels. LRRK2 kinase inhibitors did not have a major effect on TLR-stimulated cytokine levels. Results suggest that the LRRK2 G2019S mutation may potentiate inflammation following activation of TLRs. However, this was not dependent on LRRK2 kinase activity. Indeed, LRRK2 kinase inhibitors had little effect on TLR-mediated inflammation under the conditions employed in this study.

Inhibition of TLR4 prevents hippocampal hypoxic-ischemic injury by regulating ferroptosis in neonatal rats

Inflammation and cell death play important roles in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Toll-like receptor 4 (TLR4) triggers the activation of the inflammatory pathway. Ferroptosis, a newly identified type of regulated cell death, is implicated in various diseases involving neuronal injury. However, the role of ferroptosis in HIBD has not been elucidated. The objectives of this study were to explore the function and mechanism of TLR4 in neuronal ferroptosis in the context of HIBD. A neonatal rat model of hypoxia-ischemia (HI) and a cell model of oxygen-glucose deprivation (OGD) were employed. TAK-242, a TLR4-specific antagonist, was used to evaluate the effect of TLR4 on neuronal ferroptosis in vivo. A TAK-242 inhibitor and a p38 inhibitor (SB203580) were administered to HT22 hippocampal neurons to explore the association between TLR4 in inflammation and ferroptosis in vitro. The effects of TLR4 on ferroptosis were assessed by the Western blot, real-time PCR, immunofluorescence staining, cell viability and transmission electron microscopy (TEM) assays. HI insult significantly upregulated the TLR4, increased the p53 level, reduced the SLC7A11 and GPX4 levels, and caused mitochondrial damage, thereby inducing neuronal ferroptosis in the hippocampus. Inhibition of TLR4 inhibited the expression of ferroptosis-related proteins, decreased the expression of ferroptosis-related genes and the proinflammatory milieu, attenuated oxidative stress and mitochondrial injury and, finally, ameliorated the activation of hippocampal neuronal ferroptosis following HIBD. Consistent with the results of these in vivo experiments, TLR4 inhibition also attenuated OGD-induced ferroptosis by suppressing oxidative stress and p38MAPK signaling, ultimately increasing neuronal cell viability. Finally, the in vitro and in vivo results demonstrated that TAK-242 exerted neuroprotective and antiferroptotic effects by suppressing TLR4-p38 MAPK signaling. TLR4 activation induced neuronal ferroptosis following both HIBD and OGD. Inhibition of TLR4 attenuated oxidative stress-induced damage, decreased the activation of ferroptosis, and attenuated neuroinflammation following HIBD. In this study, we demonstrated that the inhibition of TLR4-p38 MAPK signaling modulates HIBD- or OGD-induced ferroptosis in neuronal cells and may play a novel role in brain homeostasis.

Lacticaseibacillus rhamnosus GG inhibits infection of human keratinocytes by Staphylococcus aureus through mechanisms involving cell surface molecules and pH reduction.

Beneficial bacteria represent an emerging tool against topical diseases, including infection caused by Staphylococcus aureus. Here, we investigated several anti-pathogenic mechanisms of the model probiotic Lacticaseibacillus rhamnosus GG against a clinical S. aureus isolate by implementing various mutants lacking important cell surface molecules. We analysed adhesion of L. rhamnosus and competitive adhesion with S. aureus to primary human keratinocytes, L. rhamnosus and S. aureus auto- and co-aggregation, S. aureus growth inhibition, keratinocyte viability increase, and monocyte Toll-like receptor (TLR) activation by L. rhamnosus as such, or with S. aureus. L. rhamnosus mutated in SpaCBA pili exhibited reduced adhesion to keratinocytes, reduced ability to prevent S. aureus adhesion to keratinocytes and reduced co-aggregation with S. aureus. Mutants in cell wall exopolysaccharides showed enhanced adhesion to keratinocytes and TLR activation in monocytes, suggesting involvement of additional cell surface molecules masked by exopolysaccharides. All L. rhamnosus strains inhibited S. aureus growth, likely due to acidification of the medium. Live (but not UV-inactivated) L. rhamnosus significantly reduced inflammatory TLR activation in monocytes by S. aureus. These data suggest the key role of SpaCBA pili and additional contribution of other cell surface molecules as well as secreted components of L. rhamnosus GG in the multifactorial inhibition of S. aureus adhesion and toxicity in the skin niche.

The role of Toll-like receptor 4 in apoptosis of brain tissue after induction of intracerebral hemorrhage

BackgroundInflammation and apoptosis caused by intracerebral hemorrhage (ICH) are two important factors that affect patient prognosis and survival. Toll-like receptor 4 (TLR4) triggers activation of the inflammatory pathway, causing synthesis and release of inflammatory factors. The inflammatory environment also causes neuronal apoptosis. However, no studies have reported the role of TLR4 in inflammation and apoptosis.MethodsWe performed survival curve analysis and behavioral scores on TLR4 knockout mice and wild-type mice after inducing ICH. We used TLR4 knockout mice and wild-type mice to make ICH models with type VII collagenase and explored the link between TLR4 in inflammation and apoptosis. We used Western blot to detect the expression of apoptosis-related proteins, inflammatory factors, and their receptors at different time points after ICH induction. The effects of TLR4 on apoptosis were observed by TUNEL, Hoechst, and HE staining techniques. The association with TLR4 in inflammation and apoptosis was explored using IL-1β and TNF-α antagonists. Data conforming to a normal distribution are expressed as mean ± standard deviation. Grade and quantitative data were compared with rank sum test and t test between two groups. P < 0.05 was considered statistically significant.ResultsTLR4 knockout significantly increased the survival rate of ICH mice. The scores of TLR4 knockout mice were significantly lower than those of wild-type mice. We found that TLR4 knockout mice significantly inhibited apoptosis and the expression of inflammatory factors after the induction of ICH. The apoptosis of ICH-induced mice was significantly improved after injecting IL-1β and TNF-α antagonists. Moreover, the anti-apoptotic effect of the antagonist in wild-type mice is more pronounced. A single injection of the antagonist failed to improve apoptosis in TLR4 knockout mice.ConclusionsWe conclude that TLR4-induced inflammation after ICH promotes neuronal apoptosis. IL-1β and TNF-α antagonists attenuate this apoptotic effect. Therefore, targeting TLR4 in patients with clinical ICH may attenuate inflammatory response, thereby attenuating apoptosis and improving prognosis.

Anti-inflammatory effects induced by ultralow concentrations of bupivacaine in combination with ultralow concentrations of sildenafil (Viagra) and vitamin D3 on inflammatory reactive brain astrocytes.

Network coupled cells, such as astrocytes, regulate their cellular homeostasis via Ca2+ signals spread between the cells through gap junctions. Intracellular Ca2+ release is controlled by different signaling pathways that can be stimulated by ATP, glutamate and serotonin (5-HT). Based on our findings, all these pathways are influenced by inflammatory agents and must be restored to fully recover the Ca2+ signaling network. An ultralow concentration of the local anesthetic agent bupivacaine reduced 5-HT-evoked intracellular Ca2+ release, and an ultralow concentration of the phosphodiesterase-5 inhibitor sildenafil in combination with vitamin D3 reduced ATP-evoked intracellular Ca2+ release. Combinations of these three substances downregulated 5-HT-, glutamate- and ATP-evoked intracellular Ca2+ release to a more normal Ca2+ signaling state. Furthermore, inflammatory Toll-like receptor 4 expression decreased with a combination of these three substances. Substance P receptor neurokinin (NK)-1 expression was reduced by ultralow concentrations of bupivacaine. Here, bupivacaine and sildenafil (at extremely low concentrations) combined with vitamin D3 have potential anti-inflammatory properties. According to the present study, drug combinations at the right concentrations, especially extremely low concentrations of bupivacaine and sildenafil, affect different cellular biochemical mechanisms and represent a potential solution for downregulating inflammatory parameters, thereby restoring cells or networks to normal physiological homeostasis.

Phytomedicine-Based Potent Antioxidant, Fisetin Protects CNS-Insult LPS-Induced Oxidative Stress-Mediated Neurodegeneration and Memory Impairment.

Phytomedicine based natural flavonoids have potent antioxidant, anti-inflammatory, and neuroprotective activities against neurodegenerative diseases. The aim of the present study is to investigate the potent neuroprotective and antioxidant potential effects of fisetin (natural flavonoid) against central nervous system (CNS)-insult, lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), neuroinflammation, neurodegeneration, and synaptic/memory deficits in adult mice. The mice were injected intraperitoneally (i.p.) with LPS (250 μg/kg/day for 1 week) and a fisetin dosage regimen (20 mg/kg/day i.p. for 2 weeks, 1 week pre-treated to LPS and 1 week co-treated with LPS). Behavioral tests, and biochemical and immunofluorescence assays were applied. Our results revealed that fisetin markedly abrogated the LPS-induced elevated ROS/oxidative stress and activated phosphorylated c-JUN N-terminal Kinase (p-JNK) in the adult mouse hippocampus. Fisetin significantly alleviated LPS-induced activated gliosis. Moreover, fisetin treatment inhibited LPS-induced activation of the inflammatory Toll-like Receptors (TLR4)/cluster of differentiation 14 (CD14)/phospho-nuclear factor kappa (NF-κB) signaling and attenuated other inflammatory mediators (tumor necrosis factor-α (TNF-α), interleukin-1 β (IL1-β), and cyclooxygenase (COX-2). Furthermore, immunoblotting and immunohistochemical results revealed that fisetin significantly reversed LPS-induced apoptotic neurodegeneration. Fisetin improved the hippocampal-dependent synaptic and memory functions in LPS-treated adult mice. In summary, our results strongly recommend that fisetin, a natural potent antioxidant, and neuroprotective phytomedicine, represents a promising, valuable, and therapeutic candidate for the prevention and treatment of neurodegenerative diseases.

Evaluation of the effects of TAK-242 and GIT-27 on methotrexate-induced liver injury

Background: Methotrexate (MTX)-induced liver injury is a common problem that is described as increased level of hepatic biomarkers that is seen in 14%–25% of patients with inflammatory bowel disease and 49% of patients with rheumatoid arthritis or as idiosyncratic induced liver injury that is seen in 1% of patients with inflammatory bowel disease, or as fibrosis and cirrhosis in 17% of rheumatoid arthritis patients and 25% of psoriatic patients. This profile may rarely progress to acute liver failure. Aim: The aim is to study the effect of TAK-242 and GIT-27 on MTX-induced liver injury. Materials and Methods: Thirty-five Albino-Wistar rats were divided into five groups: the first group was maintained on distilled water, the second group was administered intraperitoneal (I.P.) dimethyl sulfoxide followed by oral MTX, the third group was administered oral MTX, the fourth group was administered I.P. TAK-242 followed by oral MTX, and the fifth group was administered I.P. GIT-27 followed by oral MTX. Results: The significant increase in markers of hepatic function, inflammatory and oxidative stress markers, as well as severe liver histopathologic change “steatosis” induced by methotrexate were alleviated in the animals pretreated with the drugs TAK-242 and GIT-27. With significant improvement in serum level of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, interleukin-6, tumor necrosis factor-α, malondialdehyde and reduced glutathione; beside an improved histopathologic profile of moderate steatosis. Conclusion: This study suggests that both TAK-242 and GIT-27 protect against liver injury induced by MTX depending on their antagonism of the inflammatory Toll-like receptors 4 and 2/6.

Complexes assembled from myelin self-antigen and a toll-like receptor antagonist promote tolerance

Abstract Myelin, a matrix that insulates the axons of neurons, is attacked by the immune system in multiple sclerosis (MS), leading to neurodegeneration. MS treatments are currently limited because they are non-curative and broadly-acting immunosuppressants that can leave patients immunocompromised. Recent reports show co-delivery of myelin peptides (MOG) along with regulatory signals can promote tolerance to myelin rather than cause inflammation. New studies reveal inflammatory toll-like receptors (TLRs) play a significant role in driving disease in both humans and mice. Further, several reports demonstrate that regular treatment with TLR9 antagonist, GpG, attenuates disease in mouse models of MS. We hypothesized that GpG DNA and cationic MOG peptide would electrostatically self-assemble into complexes, allowing co-delivery, increased cargo protection, and enhanced uptake by immune cells. These complexes might redirect the response against myelin through suppression of inflammatory TLR9 signaling during myelin presentation. Importantly, polyplexes significantly reduced inflammatory TLR9 signaling in a reporter cell line. Primary dendritic cells (DCs) internalized MOG-GpG complexes, resulting in a reduction of activation and decreased proliferation of myelin-reactive T cells. In a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), complex injection reduced disease severity more than 2-fold and incidence by 30%. This data demonstrates the ability of GpG to condense self-antigens while remaining an active immunomodulator. This work also suggests a new carrier-free platform for co-delivery of antigens and immune signals that still provides the more attractive features of biomaterials.

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis

ABSTRACTThe Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling.

O77. Functional screening of toll-like receptors in seven trophoblast cell lines

Introduction Pregnancy is considered a natural inflammatory condition, but pregnancy complications such as preeclampsia and miscarriage are linked to excessive inflammation in the placenta. Toll-like receptors (TLRs) serve as sensors for danger signals and are crucial for initiating inflammation. A successful pregnancy depends on proper function of fetal trophoblasts, the main cell type in the placenta. We have shown broad functional TLR expression in primary first trimester trophoblasts (Tangeras et al., J. Reprod. Immunol., 2014), and trophoblast TLR activation may contribute substantially to placental inflammation. Trophoblast cell lines are commonly used as surrogates for primary trophoblasts for in vitro research. With respect to TLR mediated inflammation, the translatability of trophoblast cell lines warrants examination. Objectives This study aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines of different origins and compare to primary first trimester trophoblasts. Methods The choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P and the SV40 transformed extravillous trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts ( n =6).Gene expression of TLR1-10 was analyzed using RT-qPCR. Following specific TLR ligand activation for 24 hours, trophoblast release of interleukin (IL)-1 β , IL-6, IL-8, IL-9, IL-10, IL-12 (p70), interferon (IFN)- γ -inducible protein (IP)-10, tumor necrosis factor (TNF)- α , IFN- γ , and vascular endothelial growth factor (VEGF)-A was measured by multiplex immunoassay. Results All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. On the contrary, the SV40 transformed cell lines showed restricted TLR gene expression, and responded to activation of TLR2, TLR3 and/or TLR4 by significantly upregulated production of inflammatory cytokines such as IL-6, IL-8 and/or IFN- γ . The primary first trimester trophoblasts demonstrated both a broad TLR gene expression profile and prominent cytokine response to TLR ligand activation. Only SGHPL-5 showed a TLR activated cytokine response comparable to primary first trimester trophoblasts. Conclusion Most of the trophoblast cell lines tested showed markedly lower inflammatory TLR properties compared to primary first trimester trophoblasts, and SGHPL-5 and HTR8/SVneo were most TLR responsive. This warrants caution when translating trophoblast immune function from cell line studies.

An LXR-NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux.

LXR-cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3-LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux.

IRAK signalling in cancer.

Innate immune signalling has an essential role in inflammation, and the dysregulation of signalling components of this pathway is increasingly being recognised as an important mediator in cancer initiation and progression. In some malignancies, dysregulation of inflammatory toll-like receptor (TLR) and interleukin-1 receptor (IL1R) signalling is typified by increased NF-κB activity, and it occurs through somatic mutations, chromosomal deletions, and/or transcriptional deregulation. Interleukin-1 receptor-associated kinase (IRAK) family members are mediators of TLR/IL1R superfamily signalling, and mounting evidence implicates these kinases as viable cancer targets. Although there have been previous efforts aimed at the development of IRAK kinase inhibitors, this is currently an area of renewed interest for cancer drug development.

Abstract 2932: HIF-2 alpha is required for the tumor stemness switch of cancer cells during hypoxia/oxidative stress.

Abstract Tumor is characterized by intermittent hypoxia and re-oxygenation that generates oxidative stress. The effect of acute hypoxia microenvironmental stress on the cancer cell behavior is not clearly known. We hypothesized that hypoxia/oxidative stress could re-program cancer cells to a highly aggressive and metastatic state, we termed "tumor stemness state" (1). To test the hypothesis, we have characterized an in vitro model of acute hypoxia/re-oxygenation (2), and identified a highly tumorigenic and metastatic side-population cells (SP) phenotype from a diverse groups of established cancer cell lines including neuroblastoma, rhabdomyosarcoma and small cell lung cancer (3). These post-hypoxia SP cells (henceforth known as SPhox cells) exhibited high metastatic activity, localization in the hypoxic niche, and expressed several stemness associated genes including Nanog, Oct-4 and Bmi-1 (3). In this present study, we report further characterization of the SPhox cells including the expression of stemness associated transcription factors, expression of inflammatory cytokines and receptors, and the putative interaction of SPhox cells with bone marrow (BM) stem cell niche. First, SPhox cells expressed very high level of Nanog, Lin 28, MYC and HIF-2 proteins. Second, SPhox expressed inflammatory Toll-like receptors. Third, SPhox cells increased tolerance to ROS-inducing agents. Fourth, SPhox cells, when co-cultured with mouse BM Sca-1 cells, the CD271+ fraction, enriched in mesenchymal stem cells (4) was expanded. This expanded CD271+/Sca-1+ cells exhibited high expression of Nanog, Lin28, Oct-4, MYC and HIF-2alpha and ABCG2. Furthermore, the conditioned media obtained from the CD271+/Sca-1+/ABCG2+ cells maintained the stemness of the SPhox cells for two weeks. These results indicated the potential interaction between the SPhox and CD271/Sca-1+ cell. However, these characteristics of SPhox cells were transient, reversible, and lasted only for 3-4 weeks, indicating an underlying transient re-programming mechanism behind the SPhox phenotype. Because, recently we discovered a HIF-2alpha dependent transient reprogramming of human ES cells (5), here we investigated the potential role of HIF-2alpha in the re-programming of SP cells to SPhox cells. We found that the specific siRNA gene silencing of HIF-2alpha reduced the SP to SPhox reprogramming by sixteen-fold. Thus, here we have further characterize the tumor stemness state, including the potential role of HIF-2alpha. We suggest that this tumor stemness state could be the part of the cancer cell defense mechanism persist, and reactivate the aggressive/metastatic phenotype of cancer. 1. Das B. PhD thesis, University of Toronto, 2007 2. Das B et al. Cancer Research, 2005 3. Das B et al. Stem Cells, 2008 4. Das B et al. In Press 5. Das B et al. Stem Cells, 2012 Citation Format: Bikul Das, Reza Bayat-Mokhtari, Rika Tsuchida, Herman Yeger. HIF-2 alpha is required for the tumor stemness switch of cancer cells during hypoxia/oxidative stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2013-2932 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Measurement of LRRK2 and Ser910/935 Phosphorylated LRRK2 in Peripheral Blood Mononuclear Cells from Idiopathic Parkinson's Disease Patients

A significant number of autosomal dominantly inherited Parkinson's disease (PD) cases are due to mutations in the leucine-rich repeat kinase 2 (LRRK2) gene. In cells, these pathogenic mutations have a number of differing effects on LRRK2 enzymatic activity and protein stability. In particular, five of the six described pathogenic LRRK2 mutations ablate the constitutive phosphorylation of LRRK2 on Ser910 and Ser935, two residues required for binding of LRRK2 to 14-3-3 proteins. This suggests a potential pathogenic role for these residues. However, LRRK2 kinase inhibitors, which have shown early promise as neuroprotective agents, also ablate the phosphorylation of Ser910 and Ser935. Additionally, LRRK2 is phosphorylated on Ser910 and Ser935 following activation of the inflammatory toll-like receptor pathway and inflammatory cytokines are often increased in PD patients. Whether LRRK2 protein or phosphorylation is altered in idiopathic PD is unknown. We therefore measured LRRK2 protein and its phosporylation in peripheral blood mononuclear cells (PBMCs) from 33 idiopathic Parkinson's disease patients and 27 age-matched controls. We found no significant difference in total LRRK2 protein levels in PBMCs from PD patients compared to controls. Furthermore, total LRRK2 protein expression was not effected by age, disease duration, disease severity or levodopa medication. The amount of phosphorylation on LRRK2 at both Ser910 and Ser935 correlated highly with total LRRK2 levels and was also unchanged in PD patients. Therefore, changes in LRRK2 Ser910/Ser935 phosphorylation in PBMCs are unlikely to contribute to idiopathic Parkinson's disease or be of utility as a disease biomarker. However, the invariance of Ser910 and Ser935 phosphorylation in PD PBMC's suggests that these residues could be used as pharmacodynamic biomarkers for the effectiveness of LRRK2 kinase inhibitors in patients.

Functional Characterization of TLR4 +3725 G/C Polymorphism and Association with Protection against Overweight

Subclinical low-grade systemic inflammation has been associated with obesity, insulin resistance and metabolic syndrome (MS). Recent studies have highlighted the role of gut microbiota in these disorders. The toll-like receptor 4 (TLR4) plays a key role in the innate immune response activation. We studied two polymorphisms (+3725G/C and 11350G/C) in the 3′ untranslated region (3′UTR) of the TLR4 gene that may alter its expression and their association with metabolic disorders related to systemic inflammation. We cloned the 3′UTR into a luciferase reporter system and compared wild-type 3′UTR (WT) and +3725C variant (MUT) constructs luciferase activities. MUT construct reduced the reporter gene activity by 30% compared to WT (P = 0.0001). To evaluate the association between these polymorphisms with biochemical and clinical overweight related variables, we conducted a population cross-sectional study in 966 men of Argentine general population. Considering smoking as a confounding variable that causes systemic inflammation, we studied these possible effects in both, smokers and nonsmokers. The 11350G/C polymorphism was not detected in our sample whereas the CC genotype of +3725 polymorphism was associated with lean subjects (p = 0.011) and higher Adiponectin levels (p = 0.021). Subjects without any NCEP/ATP III MS component were associated with this genotype as well (p = 0.001). These results were strengthened in nonsmokers, in which CC genotype was associated with lean subjects (p = 0.003) and compared with G carriers showed significantly lower BMI (25.53 vs. 28.60 kg/m2; p = 0.023) and waist circumference (89.27 vs. 97.51 cm; p = 0.025). None of these associations were found in smokers. These results showed that +3725C variant has a functional effect down-regulating gene expression and it could be considered as a predictive factor against overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant could predict a lower prevalence of chronic metabolic disorders.

Decreased miR-181a Expression in Monocytes of Obese Patients Is Associated with the Occurrence of Metabolic Syndrome and Coronary Artery Disease

Inflammation during obesity is associated with higher risk of metabolic syndrome and coronary artery disease (CAD). Activation of the inflammatory toll-like receptor (TLR)/nuclear factor κB (NFκB) signaling in monocytes contributes to inflammation. Weight loss after bariatric surgery leads to significant improvement of obesity-related comorbidities. MicroRNA (miR), a class of small noncoding RNA, have been implicated as negative regulators of inflammatory processes. This study sought to identify dysregulated miR in monocytes of obese patients associated with TLR/NFκB signaling, metabolic syndrome, and CAD. This retrospective study included two independent cohorts of 21 morbidly obese and 125 high-risk obese and nonobese patients in a hospitalized care setting. INTERVENTION included bariatric surgery (n = 21) with a 3-month follow-up. miR expressions in CD14(+) monocytes were determined by microarray analysis. TLR/NFκB-related miR were identified by an in silico target prediction analysis. Their expression was validated by quantitative RT-PCR. Their association with metabolic syndrome and angiographically documented CAD was assessed. miR-181a, -181b, and -181d, identified as possible regulators of the TLR/NFκB signaling, were decreased in obese monocytes, and weight loss normalized their expression to levels observed in monocytes of lean persons. miR-181a but not miR-181b and miR-181d was associated with a higher number of metabolic syndrome components and with CAD even after adjustment for traditional risk factors, obesity and the metabolic syndrome. This study demonstrates that the TLR/NFκB-related miR-181a is down-regulated in monocytes of obese patients and suggests that it is a putative biomarker of metabolic syndrome and CAD.

ATF3 protects against atherosclerosis by suppressing 25-hydroxycholesterol–induced lipid body formation

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. We demonstrate that macrophage lipid body formation can be induced by modified lipoproteins or by inflammatory Toll-like receptor agonists. We used an unbiased approach to study the overlap in these pathways to identify regulators that control foam cell formation and atherogenesis. An analysis method integrating epigenomic and transcriptomic datasets with a transcription factor (TF) binding site prediction algorithm suggested that the TF ATF3 may regulate macrophage foam cell formation. Indeed, we found that deletion of this TF results in increased lipid body accumulation, and that ATF3 directly regulates transcription of the gene encoding cholesterol 25-hydroxylase. We further showed that production of 25-hydroxycholesterol (25-HC) promotes macrophage foam cell formation. Finally, deletion of ATF3 in Apoe(-/-) mice led to in vivo increases in foam cell formation, aortic 25-HC levels, and disease progression. These results define a previously unknown role for ATF3 in controlling macrophage lipid metabolism and demonstrate that ATF3 is a key intersection point for lipid metabolic and inflammatory pathways in these cells.

Apolipoprotein A-I Attenuates Palmitate-Mediated NF-κB Activation by Reducing Toll-Like Receptor-4 Recruitment into Lipid Rafts

While high-density lipoprotein (HDL) is known to protect against a wide range of inflammatory stimuli, its anti-inflammatory mechanisms are not well understood. Furthermore, HDL's protective effects against saturated dietary fats have not been previously described. In this study, we used endothelial cells to demonstrate that while palmitic acid activates NF-κB signaling, apolipoprotein A–I, (apoA-I), the major protein component of HDL, attenuates palmitate-induced NF-κB activation. Further, vascular NF-κB signaling (IL-6, MCP-1, TNF-α) and macrophage markers (CD68, CD11c) induced by 24 weeks of a diabetogenic diet containing cholesterol (DDC) is reduced in human apoA-I overexpressing transgenic C57BL/6 mice compared to age-matched WT controls. Moreover, WT mice on DDC compared to a chow diet display increased gene expression of lipid raft markers such as Caveolin-1 and Flotillin-1, and inflammatory Toll-like receptors (TLRs) (TLR2, TLR4) in the vasculature. However apoA-I transgenic mice on DDC show markedly reduced expression of these genes. Finally, we show that in endothelial cells TLR4 is recruited into lipid rafts in response to palmitate, and that apoA-I prevents palmitate-induced TLR4 trafficking into lipid rafts, thereby blocking NF-κB activation. Thus, apoA-I overexpression might be a useful therapeutic tool against vascular inflammation.

Mitochondrial Reactive Oxygen Species and Risk of Atherosclerosis

High levels of reactive oxygen species (ROS) are observed in chronic human diseases such as obesity, type 2 diabetes, atherosclerosis, and cardiovascular diseases. In addition to the presence of oxidative stress, these diseases are also characterized by deregulated inflammatory responses. Our first aim is to discuss distinct molecular pathways that determine the rate of mitochondrial ROS (mtROS) production and identify agents and enzymes that disrupt the balance between ROS generation and ROS elimination. Recent studies exploring the mechanisms linking ROS and inflammation found that ROS derived from mitochondria act as signal-transducing molecules that provoke endothelial dysfunction associated with uncoupling of nitric oxide synthase, induce the infiltration and activation of inflammatory cells, and increase apoptosis of endothelial and vascular smooth muscle cells. Therefore, our second aim is to give a comprehensive overview of the role of mtROS in all these processes contributing to atherosclerotic lesion progression and causing plaque erosion and rupture. Our third aim is to emphasize the role of the inflammatory toll-like receptor 2/NF-κB signaling pathway in the induction of pro-inflammatory cytokines and mtROS production in relation to insulin resistance, type 2 diabetes, and atherosclerosis. Because mtROS play an active role in several pathogenic mechanisms there is need for mitochondria-targeted antioxidants. Preliminary experiments in cell and animal models of cardiovascular diseases showed that some mitochondria-targeted antioxidants indeed reduce ROS production. However, wide-spread use in humans requires the development of specific and sensitive assays to evaluate mitochondrial oxidative stress and the development of orally active compounds.