GPR40 deficiency worsens metabolic syndrome-associated periodontitis in mice.

BACKGROUND AND OBJECTIVE: G protein-coupled receptor 40 (GPR40) is a receptor for medium- and long-chain free fatty acids (FFAs). GPR40 activation improves type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), and the complications of T2DM and MetS. Periodontitis, a common oral inflammatory disease initiated by periodontal pathogens, is another complication of T2DM and MetS. Since FFAs play a key role in the pathogenesis of MetS which exacerbates periodontal inflammation and GPR40 is a FFA receptor with anti-inflammatory properties, it is important to define the role of GPR40 in MetS-associated periodontitis. MATERIALS AND METHODS: We induced MetS and periodontitis by high-fat diet and periodontal injection of lipopolysaccharide (LPS), respectively, in wild-type and GPR40-deficient mice and determined alveolar bone loss and periodontal inflammation using micro-computed tomography, histology, and osteoclast staining. We also performed in vitro study to determine the role of GPR40 in the expression of proinflammatory genes. RESULTS: The primary outcome of the study is that GPR40 deficiency increased alveolar bone loss and enhanced osteoclastogenesis in control mice and the mice with both MetS and periodontitis. GPR40 deficiency also augmented periodontal inflammation in control mice and the mice with both MetS and periodontitis. Furthermore, GPR40 deficiency led to increased plasma lipids and insulin resistance in control mice but had no effect on the metabolic parameters in mice with MetS alone. For mice with both MetS and periodontitis, GPR40 deficiency increased insulin resistance. Finally, in vitro studies with macrophages showed that deficiency or inhibition of GPR40 upregulated proinflammatory genes while activation of GPR40 downregulated proinflammatory gene expression stimulated synergistically by LPS and palmitic acid. CONCLUSION: GPR40 deficiency worsens alveolar bone loss and periodontal inflammation in mice with both periodontitis and MetS, suggesting that GPR40 plays a favorable role in MetS-associated periodontitis. Furthermore, GPR40 deficiency or inhibition in macrophages further upregulated proinflammatory and pro-osteoclastogenic genes induced by LPS and palmitic acid, suggesting that GPR40 has anti-inflammatory and anti-osteoclastogenic properties.

High-fat diet-induced metabolic syndrome increases ligature-induced alveolar bone loss in mice.

BACKGROUND: It has been well documented that metabolic syndrome (MetS) increases severity of periodontitis. In this study, we determined the effect of high-fat diet (HFD)-induced MetS on alveolar bone loss in a mouse model with ligature-induced periodontitis. To understand how MetS increases bone loss, we tested our hypothesis that palmitic acid (PA), a most abundant saturated fatty acid in the HFD, interacts with lipopolysaccharide (LPS) to promote osteoclastogenesis. METHODS: We induced MetS by feeding mice HFD for 18 weeks and induced periodontitis with ligature placement. After treatments, we assessed alveolar bone loss using micro-computed tomography and determined osteoclastogenesis using tartrate-resistant acid phosphatase (TRAP) staining. To explore the mechanisms, we treated macrophages with PA, LPS or both and analyzed the osteoclast formation and cytokine expression in macrophages. RESULTS: While ligature robustly induced periodontitis in mice with or without MetS, the mice with MetS had more bone loss than those without MetS. PA and LPS cooperatively induced osteoclast formation and stimulated the expression of inflammatory cytokines involved in osteoclastogenesis potentially via a FAT/CD36-dependent mechanism in macrophages. CONCLUSIONS: HFD-induced MetS increases alveolar bone loss in mice with ligature-induced periodontitis, and PA and LPS cooperatively stimulate osteoclast formation and proinflammatory gene expression in macrophages.

The Presence of Periodontitis Exacerbates Non-Alcoholic Fatty Liver Disease via Sphingolipid Metabolism-Associated Insulin Resistance and Hepatic Inflammation in Mice with Metabolic Syndrome.

Clinical studies have shown that periodontitis is associated with non-alcoholic fatty liver disease (NAFLD). However, it remains unclear if periodontitis contributes to the progression of NAFLD. In this study, we generated a mouse model with high-fat diet (HFD)-induced metabolic syndrome (MetS) and NAFLD and oral P. gingivalis inoculation-induced periodontitis. Results showed that the presence of periodontitis increased insulin resistance and hepatic inflammation and exacerbated the progression of NAFLD. To determine the role of sphingolipid metabolism in the association between NAFLD and periodontitis, we also treated mice with imipramine, an inhibitor of acid sphingomyelinase (ASMase), and demonstrated that imipramine treatment significantly alleviated insulin resistance and hepatic inflammation, and improved NAFLD. Studies performed in vitro showed that lipopolysaccharide (LPS) and palmitic acid (PA), a major saturated fatty acid associated with MetS and NAFLD, synergistically increased the production of ceramide, a bioactive sphingolipid involved in NAFLD progression in macrophages but imipramine effectively reversed the ceramide production stimulated by LPS and PA. Taken together, this study showed for the first time that the presence of periodontitis contributed to the progression of NAFLD, likely due to alterations in sphingolipid metabolism that led to exacerbated insulin resistance and hepatic inflammation. This study also showed that targeting ASMase with imipramine improves NAFLD by reducing insulin resistance and hepatic inflammation.

Inhibition of acid sphingomyelinase by imipramine abolishes the synergy between metabolic syndrome and periodontitis on alveolar bone loss.

BACKGROUND AND OBJECTIVE: Clinical studies have shown that metabolic syndrome (MetS) exacerbates periodontitis. However, the underlying mechanisms remain largely unknown. Since our animal study has shown that high-fat diet-induced MetS exacerbates lipopolysaccharide (LPS)-stimulated periodontitis in mouse model and our in vitro study showed that acid sphingomyelinase (aSMase) plays a key role in the amplification of LPS-triggered pro-inflammatory response by palmitic acid (PA) in macrophages, we tested our hypothesis that inhibitor of aSMase attenuates MetS-exacerbated periodontitis in animal model. Furthermore, to explore the potential underlying mechanisms, we tested our hypothesis that aSMase inhibitor downregulates pro-inflammatory and pro-osteoclastogenic gene expression in macrophages in vitro. MATERIAL AND METHODS: We induced MetS and periodontitis in C57BL/6 mice by feeding high-fat diet (HFD) and periodontal injection of A. actinomycetemcomitans LPS, respectively, and treated mice with imipramine, a well-established inhibitor of aSMase. Micro-computed tomography (micro-CT), tartrate-resistant acid phosphatase staining, histological and pathological evaluations as well as cell cultures were performed to evaluate alveolar bone loss, osteoclast formation, periodontal inflammation and pro-inflammatory gene expression. RESULTS: Analysis of metabolic parameter showed that while HFD induced MetS by increasing bodyweight, insulin resistance, cholesterol and free fatty acids, imipramine reduced free fatty acids but had no significant effects on other metabolic parameters. MicroCT showed that either MetS or periodontitis significantly reduced bone volume fraction (BVF) of maxilla and the combination of MetS and periodontitis further reduced BVF. However, imipramine increased BVF in mice with both MetS and periodontitis to a level similar to that in mice with periodontitis alone, suggesting that imipramine abolished the synergy between MetS and periodontitis on alveolar bone loss. Consistently, results showed that imipramine inhibited osteoclast formation and periodontal inflammation in mice with both MetS and periodontitis. To elucidate the mechanisms by which imipramine attenuates MetS-exacerbated periodontitis, we showed that imipramine inhibited the upregulation of pro-inflammatory cytokines and transcription factor c-FOS as well as ceramide production by LPS plus PA in macrophages. CONCLUSION: This study has shown that imipramine as an inhibitor of aSMase abolishes the synergy between MetS and periodontitis on alveolar bone loss in animal model and inhibits pro-inflammatory and pro-osteoclastogenic gene expression in macrophages in vitro. This study provides the first evidence that aSMase is a potential therapeutic target for MetS-exacerbated periodontitis.

Neural correlates of single-vessel haemodynamic responses in vivo.

Neural activation increases blood flow locally. This vascular signal is used by functional imaging techniques to infer the location and strength of neural activity. However, the precise spatial scale over which neural and vascular signals are correlated is unknown. Furthermore, the relative role of synaptic and spiking activity in driving haemodynamic signals is controversial. Previous studies recorded local field potentials as a measure of synaptic activity together with spiking activity and low-resolution haemodynamic imaging. Here we used two-photon microscopy to measure sensory-evoked responses of individual blood vessels (dilation, blood velocity) while imaging synaptic and spiking activity in the surrounding tissue using fluorescent glutamate and calcium sensors. In cat primary visual cortex, where neurons are clustered by their preference for stimulus orientation, we discovered new maps for excitatory synaptic activity, which were organized similarly to those for spiking activity but were less selective for stimulus orientation and direction. We generated tuning curves for individual vessel responses for the first time and found that parenchymal vessels in cortical layer 2/3 were orientation selective. Neighbouring penetrating arterioles had different orientation preferences. Pial surface arteries in cats, as well as surface arteries and penetrating arterioles in rat visual cortex (where orientation maps do not exist), responded to visual stimuli but had no orientation selectivity. We integrated synaptic or spiking responses around individual parenchymal vessels in cats and established that the vascular and neural responses had the same orientation preference. However, synaptic and spiking responses were more selective than vascular responses--vessels frequently responded robustly to stimuli that evoked little to no neural activity in the surrounding tissue. Thus, local neural and haemodynamic signals were partly decoupled. Together, these results indicate that intrinsic cortical properties, such as propagation of vascular dilation between neighbouring columns, need to be accounted for when decoding haemodynamic signals.

GPR40 deficiency is associated with hepatic FAT/CD36 upregulation, steatosis, inflammation, and cell injury in C57BL/6 mice.

G-protein-coupled receptor 40 (GPR40) is highly expressed in pancreatic islets, and its activation increases glucose-stimulated insulin secretion from pancreas. Therefore, GPR40 is considered as a target for type 2 diabetes mellitus (T2DM). Since nonalcoholic fatty liver disease (NAFLD) is associated with T2DM and GPR40 is also expressed by hepatocytes and macrophages, it is important to understand the role of GPR40 in NAFLD. However, the role of GPR40 in NAFLD in animal models has not been well defined. In this study, we fed wild-type or GPR40 knockout C57BL/6 mice a high-fat diet (HFD) for 20 wk and then assessed the effect of GPR40 deficiency on HFD-induced NAFLD. Assays on metabolic parameters showed that an HFD increased body weight, glucose, insulin, insulin resistance, cholesterol, and alanine aminotransferase (ALT), and GPR40 deficiency did not mitigate the HFD-induced metabolic abnormalities. In contrast, we found that GPR40 deficiency was associated with increased body weight, insulin, insulin resistance, cholesterol, and ALT in control mice fed a low-fat diet (LFD). Surprisingly, histology and Oil Red O staining showed that GPR40 deficiency in LFD-fed mice was associated with steatosis. Immunohistochemical analysis showed that GPR40 deficiency also increased F4/80, a macrophage biomarker, in LFD-fed mice. Furthermore, results showed that GPR40 deficiency led to a robust upregulation of hepatic fatty acid translocase (FAT)/CD36 expression. Finally, our in vitro studies showed that GPR40 knockdown by siRNA or a GPR40 antagonist increased palmitic acid-induced FAT/CD36 mRNA in hepatocytes. Taken together, this study indicates that GPR40 plays an important role in homeostasis of hepatic metabolism and inflammation and inhibits nonalcoholic steatohepatitis by possible modulation of FAT/CD36 expression.

Amitriptyline inhibits nonalcoholic steatohepatitis and atherosclerosis induced by high-fat diet and LPS through modulation of sphingolipid metabolism.

We reported previously that increased acid sphingomyelinase (ASMase)-catalyzed hydrolysis of sphingomyelin, which leads to increases in ceramide and sphingosine 1 phosphate (S1P), played a key role in the synergistic upregulation of proinflammatory cytokines by palmitic acid (PA), a major saturated fatty acid, and lipopolysaccharide (LPS) in macrophages. Since macrophages are vital players in nonalcoholic steatohepatitis (NASH) and atherosclerosis, we assessed the effect of ASMase inhibition on NASH and atherosclerosis cooperatively induced by high-PA-containing high-fat diet (HP-HFD) and LPS in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice were fed HP-HFD, injected with low dose of LPS and treated with or without the ASMase inhibitor amitriptyline. The neutral sphingomyelinase inhibitor GW4869 was used as control. Metabolic study showed that both amitriptyline and GW4869 reduced glucose, lipids, and insulin resistance. Histological analysis and Oil Red O staining showed that amitriptyline robustly reduced hepatic steatosis while GW4869 had modest effects. Interestingly, immunohistochemical study showed that amitriptyline, but not GW4869, strongly reduced hepatic inflammation. Furthermore, results showed that both amitriptyline and GW4869 attenuated atherosclerosis. To elucidate the underlying mechanisms whereby amitriptyline inhibited both NASH and atherosclerosis, but GW4869 only inhibited atherosclerosis, we found that amitriptyline, but not GW4869, downregulated proinflammatory cytokines in macrophages. Finally, we found that inhibition of sphingosine 1 phosphate production is a potential mechanism whereby amitriptyline inhibited proinflammatory cytokines. Collectively, this study showed that amitriptyline inhibited NASH and atherosclerosis through modulation of sphingolipid metabolism in LDLR-/- mice, indicating that sphingolipid metabolism in macrophages plays a crucial role in the linkage of NASH and atherosclerosis.

Acid sphingomyelinase deficiency exacerbates LPS-induced experimental periodontitis.

BACKGROUND: Mutation of the gene for acid sphingomyelinase (ASMase) causes Niemann-Pick disease. However, the effect of ASMase deficiency on periodontal health is unknown. Periodontal disease is a disease resulting from infection and inflammation of periodontal tissue and alveolar bone that support the teeth. The goal of this study was to determine the role of ASMase deficiency in periodontal inflammation and alveolar bone loss. METHODS: We induced periodontitis in wild-type and ASMase-deficient (ASMase-/- ) mice with periodontal lipopolysaccharide (LPS) injection and compared the alveolar bone loss and periodontal inflammation between these mice. RESULTS: Results showed that ASMase deficiency did not significantly change metabolic parameters, but exacerbated LPS-induced alveolar bone loss, osteoclastogenesis, and periodontal tissue inflammation. To understand the mechanisms by which ASMase deficiency aggravates LPS-induced periodontitis, we analyzed sphingolipids in periodontal tissues. Results showed that ASMase deficiency led to increases in not only sphingomyelin, but also ceramide (CER), a bioactive sphingolipid known to promote inflammation. Results further showed that ASMase deficiency increased CER de novo synthesis. CONCLUSION: ASMase deficiency exacerbated LPS-induced alveolar bone loss and periodontal inflammation. ASMase deficiency leads to an unexpected CER increase by stimulating de novo synthesis CER, which is likely to be involved in the ASMase deficiency-exacerbated periodontitis.

Interaction of palmitate and LPS regulates cytokine expression and apoptosis through sphingolipids in human retinal microvascular endothelial cells.

Studies have implicated saturated fatty acid (SFA) and lipopolysaccharide (LPS) in diabetic retinopathy. Since type 2 diabetes is associated with increases in both SFA and LPS in circulation, we investigated how SFA interacts with LPS to regulate proinflammatory cytokine expression and apoptosis in human retinal microvascular endothelial cells (HRMVECs) and the underlying mechanisms. HRMVECs were challenged with palmitate, a major SFA, LPS or palmitate plus LPS and the expression of proinflammatory cytokines were quantified using real-time PCR and enzyme-linked immunosorbent assay. The interaction between palmitate and LPS on inflammatory signaling and sphingolipid metabolism was demonstrated by immunoblotting and lipidomic analysis, respectively. The effect of palmitate and LPS on apoptosis was also studied by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and histone-associated DNA fragment assays. Results showed that palmitate robustly stimulated the expression of proinflammatory cytokines including interleukin (IL)-6 and IL-1ß, and the combination of palmitate and LPS further upregulated the proinflammatory cytokines by cooperatively stimulating inflammatory signaling pathways. Results also showed that while palmitate stimulated ceramide (CER) production via CER de novo synthesis and sphingomyelin (SM) hydrolysis, addition of LPS further increased CER de novo synthesis, but not SM hydrolysis. The involvement of sphingolipids in the cooperative stimulation by palmitate and LPS on cytokine expression was indicated by the findings that the inhibitor of CER de novo synthesis or SM hydrolysis attenuated the stimulation of IL-6 expression by palmitate and LPS. In addition, our study showed that fatty acid receptors GPR40 and CD36 were involved in the IL-6 upregulation by palmitate and LPS. Furthermore, palmitate induced apoptosis via CER production, but addition of LPS did not further increase apoptosis. Taken together, this study showed that palmitate interacted with LPS to upregulate cytokine expression via free fatty acid receptor-mediated inflammatory signaling and sphingolipid metabolism in HRMVECs. In contrast, the interaction between palmitate and LPS did not further increase apoptosis.

Upregulation of free fatty acid receptors in periodontal tissues of patients with metabolic syndrome and periodontitis.

BACKGROUND AND OBJECTIVE: Metabolic syndrome (MetS) exacerbates periodontitis. Since saturated fatty acid (SFA) is increased in MetS and enhances lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in macrophages, it has been considered to play a role in MetS-exacerbated periodontitis. However, it remains unknown how fatty acid receptors, which mediate the interaction of cells with SFA and uptake of SFA, are expressed and regulated in the periodontal tissue. In this study, we tested our hypothesis that the periodontal expression of fatty acid receptors GPR40 and CD36 is increased in patients with both MetS and periodontitis. We also determined the effect of SFA and LPS on GPR40 and CD36 expression in vitro. MATERIAL AND METHODS: Periodontal tissue specimens were collected from 11 participants without MetS and periodontitis, 12 participants with MetS, 11 participants with periodontitis, and 14 participants with both MetS and periodontitis after surgeries. The tissues were processed, and GPR40 and CD36 were detected by immunohistochemistry. Furthermore, cultured macrophages and gingival fibroblasts were treated with LPS, palmitate, a major SFA, or LPS plus palmitate and the expression of GPR40 and CD36 was then quantified. RESULTS: Analysis of clinical data showed that age, smoker, gender, and race/ethnicity were not significantly different among 4 groups. Immunohistochemistry showed that GPR40 and CD36 were expressed by epithelial cells, fibroblasts, and immune cells. Quantitative data showed that GPR40 expression is increased in patients with periodontitis, MetS, or both periodontitis and MetS while CD36 expression is increased only in patients with both periodontitis and MetS. The in vitro studies showed that the expression of GPR40 and CD36 in macrophages and fibroblasts was upregulated by the combination of LPS and palmitate. CONCLUSION: Periodontal expression of GPR40 and CD36 was upregulated in patients with both MetS and periodontitis, and GPR40 and CD36 in macrophages and fibroblasts were upregulated in vitro by the combination of LPS and palmitate, suggesting that GPR40 and CD36 may be involved in MetS-exacerbated periodontitis.

Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and consumption of high-fat diet (HFD) is a risk factor for NAFLD. The HFD not only increases intake of saturated fatty acid (SFA) but also induces metabolic endotoxemia, an HFD-associated increase in circulating lipopolysaccharide (LPS). Although it is known that SFA or LPS promote hepatic inflammation, a hallmark of NAFLD, it remains unclear how SFA in combination with LPS stimulates host inflammatory response in hepatocytes. In this study, we performed both in vivo and in vitro experiments to investigate the effect of SFA in combination with LPS on proinflammatory gene expression in hepatocytes. Our animal study showed that feeding low-density lipoprotein-deficient mice HFD enriched with SFA and injection of low-dose LPS cooperatively stimulated IL-6 expression in livers. To understand how SFA and LPS interact to promote IL-6 expression, our in vitro studies showed that palmitic acid (PA), a major SFA, and LPS exerted synergistic effect on the expression of IL-6 in hepatocytes. Furthermore, coculture of hepatocytes with macrophages resulted in a greater IL-6 expression than culture of hepatocytes without macrophages in response to the combination of PA and LPS. Finally, we observed that LPS and PA increased ceramide production by cooperatively stimulating ceramide de novo synthesis, which played an essential role in the synergistic stimulation of proinflammatory gene expression by LPS and PA. Taken together, this study showed that SFA in combination with LPS stimulated a strong inflammatory response in hepatocytes in vivo and in vitro.

F(ab')2 fragments of anti-oxidized LDL IgG attenuate vascular inflammation and atherogenesis in diabetic LDL receptor-deficient mice.

Considerable evidence is available supporting the atherogenic role of immune complexes (IC) formed by modified forms of LDL and their corresponding antibodies in humans and other species. In this study, we assessed the effect of IgG F(ab')2 fragments of murine anti-mouse oxLDL, which binds oxLDL forming IC that cannot interact with Fcγ receptors, on the development of atherosclerosis in diabetic LDL receptor-deficient (LDLR-/-) mice. Immunohistochemical study showed that treatment with the F(ab')2 fragments for 8weeks significantly reduced the content of macrophages and interleukin 6 expression in atherosclerotic lesions. Furthermore, histological study showed that treatment with the same F(ab')2 fragments significantly reduced atherosclerotic lesions in diabetic LDLR-/- mice. Taken together, this study demonstrated for the first time that F(ab')2 fragments of anti-oxLDL IgG inhibited vascular inflammation and atherogenesis in diabetic LDLR-/- mice and uncovered a possible new avenue for therapy in patients at high risk to progress to cardiovascular complications.

The shape of dendritic arbors in different functional domains of the cortical orientation map.

The neocortex is organized into macroscopic functional maps. However, at the microscopic scale, the functional preference and degree of feature selectivity between neighboring neurons can vary considerably. In the primary visual cortex, adjacent neurons in iso-orientation domains share the same orientation preference, whereas neighboring neurons near pinwheel centers are tuned to different stimulus orientations. Moreover, several studies have found greater orientation selectivity in iso-orientation domains than in pinwheel centers. These differences suggest that neurons sample local inputs in a spatially homogenous fashion and independently of the location of their soma on the orientation map. Here we determine whether dendritic geometry is affected by neuronal position on the orientation map. We labeled individual layer 2/3 pyramidal neurons with fluorescent dyes in specific domains of the orientation map in cat primary visual cortex and imaged their dendritic trees in vivo by two-photon microscopy. We found that the circularity and uniformity of dendritic trees is independent of somatic position on the orientation map. Moreover, the dendrites of neurons located close to pinwheel centers extend across all orientation domains in an unbiased fashion. Thus, unbiased dendritic trees appear to provide an anatomical substrate for the systematic variations in feature selectivity across the orientation map.

An artery-specific fluorescent dye for studying neurovascular coupling.

We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus-evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.

Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target.

Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.

The Endogenous Expression of BMI1 in Adult Human Eyes.

BMI1, also known as B lymphoma Mo-MLV insertion region 1, is a protein in the Polycomb group that is implicated in various cellular processes, including stem cell self-renewal and the regulation of cellular senescence. BMI1 plays a role in the regulation of retinal progenitor cells and the renewal of adult neuronal cells. However, the presence, location, and quantification of BMI1 in the adult human eye have never previously been reported. In this study, we collected 45 frozen globes from eye banks, and ocular tissues were dissected. Protein was quantified by utilizing a custom electrochemiluminescence (ECL) assay developed to quantify the BMI1 protein. BMI1 was found in all ocular tissues at the following levels: the retina (1483.6 ± 191.7 pg/mL) and the RPE (296.4 ± 78.1 pg/mL). BMI1 expression was noted ubiquitously in the GCL (ganglion cell layer), the INL (inner nuclear layer), the ONL (outer nuclear layer), and the RPE (retinal pigment epithelium) via immunofluorescence, with higher levels in the inner than in the outer retinal layers and the RPE. These data confirm that BMI1 is expressed in the human retina. Further studies will illuminate the role that BMI1 plays in ocular cells. BMI1 levels are lower in aged retinas, possibly reflecting changes in retinal somatic and stem cell maintenance and disease susceptibility.

FLI1 in PBMCs contributes to elevated inflammation in combat-related posttraumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with significant public health implications that arise following exposure to traumatic events. Recent studies highlight the involvement of immune dysregulation in PTSD, characterized by elevated inflammatory markers. However, the precise mechanisms underlying this immune imbalance remain unclear. Previous research has implicated friend leukemia virus integration 1 (FLI1), an erythroblast transformation-specific (ETS) transcription factor, in inflammatory responses in sepsis and Alzheimer's disease. Elevated FLI1 levels in peripheral blood mononuclear cells (PBMCs) have been linked to lupus severity. Yet, FLI1's role in PTSD-related inflammation remains unexplored. In our study, PBMCs were collected from Veterans with and without PTSD. We found significantly increased FLI1 expression in PBMCs from PTSD-afflicted Veterans, particularly in CD4+ T cells, with no notable changes in CD8+ T cells. Stimulation with LPS led to heightened FLI1 expression and elevated levels of inflammatory cytokines IL-6 and IFNγ in PTSD PBMCs compared to controls. Knockdown of FLI1 using Gapmers in PTSD PBMCs resulted in a marked reduction in inflammatory cytokine levels, restoring them to control group levels. Additionally, co-culturing PBMCs from both control and PTSD Veterans with the human brain microglia cell line HMC3 revealed increased inflammatory mediator levels in HMC3. Remarkably, HMC3 cells co-cultured with PTSD PBMCs treated with FLI1 Gapmers exhibited significantly lower inflammatory mediator levels compared to control Gapmer-treated PTSD PBMCs. These findings suggest that suppressing FLI1 may rebalance immune activity in PBMCs and mitigate microglial activation in the brain. Such insights could provide novel therapeutic strategies for PTSD.

MD-2 is involved in the stimulation of matrix metalloproteinase-1 expression by interferon-γ and high glucose in mononuclear cells - a potential role of MD-2 in Toll-like receptor 4-independent signalling.

We reported recently that treatment of diabetic apolipoprotein E-deficient mice with the Toll-like receptor 4 (TLR4) antagonist Rs-LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs-LPS antagonizes TLR4 by targeting TLR4 co-receptor MD-2, this finding indicates that MD-2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD-2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon-γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD-2 is involved in IFN-γ and high-glucose-regulated gene expression in mononuclear cells. Results showed that IFN-γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP-1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs-LPS inhibited the MMP-1 stimulation. To provide more evidence for a role of MD-2 in IFN-γ-stimulated MMP-1, studies using antibodies and small interfering RNA demonstrated that MD-2 blockade or knockdown attenuated the effect of IFN-γ on MMP-1. Furthermore, studies using PCR arrays showed that MD-2 blockade had a similar effect as IFN-γ receptor blockade on the inhibition of IFN-γ-stimulated pro-inflammatory molecules. Although these findings indicate the involvement of MD-2 in IFN-γ signalling, we also observed that MD-2 was up-regulated by IFN-γ and high glucose. We found that MD-2 up-regulation by IFN-γ played an essential role in the synergistic effect of IFN-γ and LPS on MMP-1 expression. Taken together, these findings indicate that MD-2 is involved in IFN-γ signalling and IFN-γ-augmented MMP-1 up-regulation by LPS.

Acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages.

Periodontal disease is more prevalent and severe in patients with diabetes than in nondiabetic patients. In addition to diabetes, a large number of studies have demonstrated an association between obesity and chronic periodontal disease. However, the underlying mechanisms have not been well understood. Since plasma free fatty acids (FAs) are elevated in obese patients and saturated FAs such as palmitic acid (PA) have been shown to increase host inflammatory response, we sought to find out how PA interacts with lipopolysaccharide (LPS), an important pathological factor involved in periodontal disease, to enhance inflammation. We found that whereas low concentration of LPS (1 ng/ml) stimulated interleukin (IL)-6 expression in RAW 264.7 macrophages, PA further augmented it fourfold. Besides IL-6, PA amplified the stimulatory effect of LPS on a large amount of Toll-like receptor (TLR)4-mediated expression of proinflammatory signaling molecules such as IL-1 receptor-associated kinase-like 2 and proinflammatory molecules, including monocyte chemotactic protein-1 and colony-stimulating factor. We also observed that PA augmented TLR4 but not TLR2 signal, and the augmentation was mediated by nuclear factor-κB (NF-κB) pathways. To further elucidate the regulatory mechanism whereby PA amplifies LPS signal, our studies showed that PA and LPS synergistically increased hydrolysis of sphingomyelin by stimulating acid sphingomyelinase (ASMase) activity, which contributed to a marked increase in ceramide production and IL-6 upregulation. Taken together, this study has demonstrated that PA markedly augments TLR4-mediated proinflammatory signaling triggered by low concentration of LPS in macrophages, and ASMase plays a key role in the augmentation.

Toll-like receptor 4 activation in microvascular endothelial cells triggers a robust inflammatory response and cross talk with mononuclear cells via interleukin-6.

OBJECTIVE: It is known that toll-like receptor 4 (TLR4) plays an important role in atherosclerosis. Because both microvascular (MIC) and macrovascular (MAC) endothelial cells (ECs) are present in atherosclerotic lesions, the present study compared TLR4-triggered inflammatory response and cross talk with mononuclear cells between MIC and MAC ECs. METHODS AND RESULTS: ELISA, real-time polymerase chain reaction, and gene expression profiling showed that TLR4 activation by lipopolysaccharide stimulated a much higher expression of inflammatory genes including cytokines, chemokines, growth factors, and adhesion molecules in MIC ECs than MAC ECs. Furthermore, coculture studies showed that TLR4 activation in MIC ECs, but not MAC ECs, induced a cross talk with U937 mononuclear cells through MIC EC-released interleukin-6 to upregulate matrix metalloproteinase-1 expression in U937 cells. To explore molecular mechanisms underlying the different responses to TLR4 activation between MIC and MAC ECs, we showed that MIC ECs had a higher expression of TLR4 and CD14 and a higher TLR4-mediated nuclear factor-kappaB activity than MAC ECs. CONCLUSIONS: The present study showed that TLR4 activation triggers a more robust inflammatory response in MIC ECs than MAC ECs. Given the importance of inflammatory cytokines and matrix metalloproteinases in plaque rupture, MIC ECs may play a key role in plaque destabilization through a TLR4-dependent mechanism.