Des-Arg(9) bradykinin as a causal metabolite for autism spectrum disorder.

BACKGROUND: Early diagnosis and therapeutic interventions can greatly enhance the developmental trajectory of children with autism spectrum disorder (ASD). However, the etiology of ASD is not completely understood. The presence of confounding factors from environment and genetics has increased the difficulty of the identification of diagnostic biomarkers for ASD. AIM: To estimate and interpret the causal relationship between ASD and metabolite profile, taking into consideration both genetic and environmental influences. METHODS: A two-sample Mendelian randomization (MR) analysis was conducted using summarized data from large-scale genome-wide association studies (GWAS) including a metabolite GWAS dataset covering 453 metabolites from 7824 European and an ASD GWAS dataset comprising 18381 ASD cases and 27969 healthy controls. Metabolites in plasma were set as exposures with ASD as the main outcome. The causal relationships were estimated using the inverse variant weight (IVW) algorithm. We also performed leave-one-out sensitivity tests to validate the robustness of the results. Based on the drafted metabolites, enrichment analysis was conducted to interpret the association via constructing a protein-protein interaction network with multi-scale evidence from databases including Infinome, SwissTargetPrediction, STRING, and Metascape. RESULTS: Des-Arg(9)-bradykinin was identified as a causal metabolite that increases the risk of ASD (ß = 0.262, SE = 0.064, PIVW = 4.64 × 10-5). The association was robust, with no significant heterogeneity among instrument variables (PMR Egger = 0.663, PIVW = 0.906) and no evidence of pleiotropy (P = 0.949). Neuroinflammation and the response to stimulus were suggested as potential biological processes mediating the association between Des-Arg(9) bradykinin and ASD. CONCLUSION: Through the application of MR, this study provides practical insights into the potential causal association between plasma metabolites and ASD. These findings offer perspectives for the discovery of diagnostic or predictive biomarkers to support clinical practice in treating ASD.

Mini review: STING activation during non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic diseases serving as a major threat to human health. While the pathogenesis of NAFLD is multi-factorial, inflammation is considered a critical factor driving the development and progression of NAFLD phenotype, including liver fibrosis. As an essential mediator of innate immunity, stimulator of interferon genes (STING) functions to promote anti-viral immunity. Accumulating evidence also indicates that STING functions to promote the proinflammatory activation of several types of liver cells, especially macrophages/Kupffer cells, in a manner independent of interferon production. Over the past several years, a significant body of literature has validated a detrimental role for STING in regulating the pathogenesis of hepatic steatosis and inflammation. In particular, the STING in macrophages/Kupffer cells has attracted much attention due to its importance in not only enhancing macrophage proinflammatory activation, but also generating macrophage-derived mediators to increase hepatocyte fat deposition and proinflammatory responses, and to activate hepatic stellate cell fibrogenic activation. Both intracellular and extracellular signals are participating in STING activation in macrophages, thereby critically contributing to NAFLD phenotype. This mini review summarizes recent advances on how STING is activated in macrophages in the context of NAFLD pathophysiology.

Hepatocyte Adenosine Kinase Promotes Excessive Fat Deposition and Liver Inflammation.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is highly associated with obesity and progresses to nonalcoholic steatohepatitis when the liver develops overt inflammatory damage. While removing adenosine in the purine salvage pathway, adenosine kinase (ADK) regulates methylation reactions. We aimed to study whether hepatocyte ADK functions as an obesogenic gene/enzyme to promote excessive fat deposition and liver inflammation. METHODS: Liver sections of human subjects were examined for ADK expression using immunohistochemistry. Mice with hepatocyte-specific ADK disruption or overexpression were examined for hepatic fat deposition and inflammation. Liver lipidomics, hepatocyte RNA sequencing (RNA-seq), and single-cell RNA-seq for liver nonparenchymal cells were performed to analyze ADK regulation of hepatocyte metabolic responses and hepatocyte-nonparenchymal cells crosstalk. RESULTS: Whereas patients with nonalcoholic fatty liver disease had increased hepatic ADK levels, mice with hepatocyte-specific ADK disruption displayed decreased hepatic fat deposition on a chow diet and were protected from diet-induced excessive hepatic fat deposition and inflammation. In contrast, mice with hepatocyte-specific ADK overexpression displayed increased body weight and adiposity and elevated degrees of hepatic steatosis and inflammation compared with control mice. RNA-seq and epigenetic analyses indicated that ADK increased hepatic DNA methylation and decreased hepatic Ppara expression and fatty acid oxidation. Lipidomic and single-cell RNA-seq analyses indicated that ADK-driven hepatocyte factors, due to mitochondrial dysfunction, enhanced macrophage proinflammatory activation in manners involving increased expression of stimulator of interferon genes. CONCLUSIONS: Hepatocyte ADK functions to promote excessive fat deposition and liver inflammation through suppressing hepatocyte fatty acid oxidation and producing hepatocyte-derived proinflammatory mediators. Therefore, hepatocyte ADK is a therapeutic target for managing obesity and nonalcoholic fatty liver disease.

Myokine myostatin is a novel predictor of one-year radiographic progression in patients with rheumatoid arthritis: A prospective cohort study.

Background: Associations between rheumatoid arthritis (RA) and reduced skeletal muscle have been studied, and we firstly reported myopenia independently predict one-year radiographic progression in RA. Myokine myostatin can negatively regulate skeletal muscle mass and promote osteoclast differentiation. However, there is no report about their relationships in RA patients. We firstly explored the relationship of serum myostatin and disease characteristics, as well as aggravated joint destruction during one-year follow-up. Methods: Consecutive RA patients were recruited from a real-world prospective cohort and completed at least one-year follow-up. Baseline serum level of myostatin was measured by enzyme-linked immunosorbent assay. Clinical data in RA patients as well as muscle index in both RA patients and healthy controls were collected. One-year radiographic progression as primary outcome was defined by a change in the total Sharp/van der Heijde modified score ≥0.5 units. Results: Totally 344 RA patients (age 47.9 ± 12.5 years, 84.0% female) and 118 healthy control subjects (age 42.8 ± 11.3 years, 74.6% female) were recruited. Compared with healthy controls, RA patients showed a higher level of serum myostatin at baseline (3.241 ± 1.679 ng/ml vs. 1.717 ± 0.872 ng/ml, P<0.001), although lower appendicular skeletal muscle mass index (ASMI, 6.0 ± 0.9 kg/m2 vs. 6.5 ± 1.0 kg/m2, P<0.001). In RA patients, those with high myostatin level showed a higher rate of radiographic progression than low myostatin group (45.3% vs. 18.6%, P<0.001). Furtherly, RA patients were stratified into four subgroups according to serum myostatin and myopenia. Compared with other three subgroups, RA patients with high myostatin overlapping myopenia had the highest rate of radiographic progression (67.2% vs. 10.3%-31.4%, P<0.001), as well as the lowest proportion of remission and the highest rate of physical dysfunction during one-year follow-up. After adjustment for confounding factors, high serum myostatin (AOR=3.451, 95%CI: 2.016-5.905) and myopenia (AOR=2.387, 95%CI: 1.416-4.022) at baseline were risk factors for one-year radiographic progression, especially for those with high myostatin overlapping myopenia (AOR=10.425, 95%CI: 3.959-27.450) as the highest-risk individuals among four subgroups. Significant synergistic interaction effect was observed between high myostatin and myopenia on one-year radiographic progression (AP=66.3%, 95%CI: 43.2%-89.3%). Conclusion: Myostatin is a novel predictor of aggravated joint destruction in RA patients which has synergistic interaction with myopenia for predicting value.

Indole supplementation ameliorates MCD-induced NASH in mice.

Indole is a microbiota metabolite that functions to protect against obesity-associated non-alcoholic fatty liver disease. The present study examined the extent to which indole supplementation alleviates the severity of non-alcoholic steatohepatitis (NASH), which is the advanced form of non-alcoholic fatty liver disease. In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregations of macrophages in the liver compared with control diet-fed mice. Upon indole supplementation, the severity of MCD-induced hepatic steatosis and inflammation, as well as liver fibrosis, was significantly decreased compared with that of MCD-fed and control-treated mice. In vitro, indole treatment caused significant decreases in lipopolysaccharide-induced proinflammatory responses in hepatocytes incubated with either basal or MCD-mimicking media. However, indole treatment only significantly decreased lipopolysaccharide-induced proinflammatory responses in bone marrow-derived macrophages incubated with basal, but not MCD-mimicking media. These differential effects suggest that, relative to the responses of macrophages to indole, the responses of hepatocytes to indole appeared to make a greater contribution to indole alleviation of NASH, in particular liver inflammation. While indole supplementation decreased liver expression of desmin in MCD-fed mice, treatment of LX2 cells (a line of hepatic stellate cells) with indole also decreased the expression of various markers of hepatic stellate cell fibrogenic activation. Lastly, indole supplementation decreased intestinal inflammation in MCD-fed mice, suggesting that decreased intestinal inflammation also was involved in indole alleviation of NASH. Collectively, these results demonstrate that indole supplementation alleviates MCD-induced NASH, which is attributable to, in large part, indole suppression of hepatocyte proinflammatory responses and hepatic stellate cell fibrogenic activation, as well as intestinal proinflammatory responses.

Recent Advances in Adipose Tissue Dysfunction and Its Role in the Pathogenesis of Non-Alcoholic Fatty Liver Disease.

Obesity is a serious ongoing health problem that significantly increases the incidence of nonalcoholic fatty liver disease (NAFLD). During obesity, adipose tissue dysfunction is obvious and characterized by increased fat deposition (adiposity) and chronic low-grade inflammation. The latter has been implicated to critically promote the development and progression of NAFLD, whose advanced form non-alcoholic steatohepatitis (NASH) is considered one of the most common causes of terminal liver diseases. This review summarizes the current knowledge on obesity-related adipose dysfunction and its roles in the pathogenesis of hepatic steatosis and inflammation, as well as liver fibrosis. A better understanding of the crosstalk between adipose tissue and liver under obesity is essential for the development of new and improved preventive and/or therapeutic approaches for managing NAFLD.

Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Steatohepatitis Is Associated with Increased Intestinal Inflammation.

Inflammation drives the pathogenesis of nonalcoholic steatohepatitis (NASH). The current study examined changes in intestinal inflammation during NASH. In male C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in severe hepatic steatosis and inflammation relative to feeding a chow diet (CD). MCD-fed mice exhibited characteristics of mucosal and submucosal inflammatory responses compared with CD-fed mice. Moreover, intestinal phosphorylation states of c-Jun N-terminal protein kinase p46 and mRNA levels of IL-1B, IL-6, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 were significantly higher and intestinal mRNA levels of IL-4 and IL-13 were significantly lower in MCD-fed mice compared with those in CD mice. Surprisingly, upon treatment with MCD-mimicking media, the proinflammatory responses in cultured intestinal epithelial CMT-93 cells did not differ significantly from those in CMT-93 cells treated with control media. In contrast, in RAW264.7 macrophages, MCD-mimicking media significantly increased the phosphorylation states of c-Jun N-terminal protein kinase p46 and mitogen-activated protein kinases p38 and mRNA levels of IL-1B, IL-6, IL-10, and tumor necrosis factor alpha under either basal or lipopolysaccharide-stimulated conditions. Collectively, these results suggest that increased intestinal inflammation is associated with NASH phenotype. Thus, elevated proinflammatory responses in macrophages likely contribute to, in large part, increased intestinal inflammation in NASH.

Adipocyte inducible 6-phosphofructo-2-kinase suppresses adipose tissue inflammation and promotes macrophage anti-inflammatory activation.

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance. To better understand how adipocytes regulate WAT inflammation, the present study generated chimeric mice in which inducible 6-phosphofructo-2-kinase was low, normal, or high in WAT while the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3) was normal in hematopoietic cells, and analyzed changes in high-fat diet (HFD)-induced WAT inflammation and systemic insulin resistance in the mice. Indicated by proinflammatory signaling and cytokine expression, the severity of HFD-induced WAT inflammation in WT â†’ Pfkfb3+/- mice, whose Pfkfb3 was disrupted in WAT adipocytes but not hematopoietic cells, was comparable with that in WT â†’ WT mice, whose Pfkfb3 was normal in all cells. In contrast, the severity of HFD-induced WAT inflammation in WT â†’ Adi-Tg mice, whose Pfkfb3 was over-expressed in WAT adipocytes but not hematopoietic cells, remained much lower than that in WT â†’ WT mice. Additionally, HFD-induced insulin resistance was correlated with the status of WAT inflammation and comparable between WT â†’ Pfkfb3+/- mice and WT â†’ WT mice, but was significantly lower in WT â†’ Adi-Tg mice than in WT â†’ WT mice. In vitro, palmitoleate decreased macrophage phosphorylation states of Jnk p46 and Nfkb p65 and potentiated the effect of interleukin 4 on suppressing macrophage proinflammatory activation. Taken together, these results suggest that the Pfkfb3 in adipocytes functions to suppress WAT inflammation. Moreover, the role played by adipocyte Pfkfb3 is attributable to, at least in part, palmitoleate promotion of macrophage anti-inflammatory activation.

Efficient and accurate identification of ear diseases using an ensemble deep learning model.

Early detection and appropriate medical treatment are of great use for ear disease. However, a new diagnostic strategy is necessary for the absence of experts and relatively low diagnostic accuracy, in which deep learning plays an important role. This paper puts forward a mechanic learning model which uses abundant otoscope image data gained in clinical cases to achieve an automatic diagnosis of ear diseases in real time. A total of 20,542 endoscopic images were employed to train nine common deep convolution neural networks. According to the characteristics of the eardrum and external auditory canal, eight kinds of ear diseases were classified, involving the majority of ear diseases, such as normal, Cholestestoma of the middle ear, Chronic suppurative otitis media, External auditory cana bleeding, Impacted cerumen, Otomycosis external, Secretory otitis media, Tympanic membrane calcification. After we evaluate these optimization schemes, two best performance models are selected to combine the ensemble classifiers with real-time automatic classification. Based on accuracy and training time, we choose a transferring learning model based on DensNet-BC169 and DensNet-BC1615, getting a result that each model has obvious improvement by using these two ensemble classifiers, and has an average accuracy of 95.59%. Considering the dependence of classifier performance on data size in transfer learning, we evaluate the high accuracy of the current model that can be attributed to large databases. Current studies are unparalleled regarding disease diversity and diagnostic precision. The real-time classifier trains the data under different acquisition conditions, which is suitable for real cases. According to this study, in the clinical case, the deep learning model is of great use in the early detection and remedy of ear diseases.

Silencing XIST mitigated lipopolysaccharide (LPS)-induced inflammatory injury in human lung fibroblast WI-38 cells through modulating miR-30b-5p/CCL16 axis and TLR4/NF-κB signaling pathway.

BACKGROUND: Emerging evidence shows that long noncoding RNA (lncRNA) has been a novel insight in various diseases, including pneumonia. Even though lncRNA X-inactive-specific transcript (XIST) is well studied, its role in pneumonia remains to be largely unrevealed. METHODS: Expression of XIST, miRNA-30b-5p (miR-30b-5p), and CC chemokine ligand 16 (CCL16) was detected using reverse transcriptase quantitative polymerase chain reaction and western blotting; their interaction was confirmed by dual-luciferase reporter assay. Apoptosis, inflammation, and toll-like receptor 4 (TLR4)/NF-κB signaling pathway were measured using methyl thiazolyl tetrazolium assay, flow cytometry, western blotting, and enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide (LPS) stimulation decreased cell viability and B cell lymphoma (Bcl)-2 expression, and increased cell apoptosis rate and expression of Bcl-2-associated X protein (Bax), cleaved-caspase-3, interleukin (IL)-6, IL-1ß, and tumor necrosis factor α (TNF-α) in WI-38 cells. Expression of XIST and CCL16 was upregulated in the serum of patients with pneumonia and LPS-induced WI-38 cells, respectively; silencing XIST and CCL16 could suppress LPS-induced apoptosis and inflammation in WI-38 cells, and this protection was abolished by miR-30b-5p downregulation. Moreover, XIST and CCL16 could physically bind to miR-30b-5p, and XIST regulated CCL16 expression via sponging miR-30b-5p. TLR4 and phosphorylated P65 (p-P65) and p-IκB-α were highly induced by LPS treatment, and this upregulation was diminished by blocking XIST, accompanied with CCL16 downregulation and miR-30b-5p upregulation. CONCLUSIONS: Silencing XIST could alleviate LPS-induced inflammatory injury in human lung fibroblast WI-38 cells through modulating miR-30b-5p/CCL16 axis and inhibiting TLR4/NF-κB signaling pathway.

Adipose tissue inflammation and systemic insulin resistance in mice with diet-induced obesity is possibly associated with disruption of PFKFB3 in hematopoietic cells.

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance; however, precisely how immune cells regulate WAT inflammation in relation to systemic insulin resistance remains to be elucidated. The present study examined a role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in hematopoietic cells in regulating WAT inflammation and systemic insulin sensitivity. Male C57BL/6J mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 12 weeks and examined for WAT inducible 6-phosphofructo-2-kinase (iPFK2) content, while additional HFD-fed mice were treated with rosiglitazone and examined for PFKFB3 mRNAs in WAT stromal vascular cells (SVC). Also, chimeric mice in which PFKFB3 was disrupted only in hematopoietic cells and control chimeric mice were also fed an HFD and examined for HFD-induced WAT inflammation and systemic insulin resistance. In vitro, adipocytes were co-cultured with bone marrow-derived macrophages and examined for adipocyte proinflammatory responses and insulin signaling. Compared with their respective levels in controls, WAT iPFK2 amount in HFD-fed mice and WAT SVC PFKFB3 mRNAs in rosiglitazone-treated mice were significantly increased. When the inflammatory responses were analyzed, peritoneal macrophages from PFKFB3-disrputed mice revealed increased proinflammatory activation and decreased anti-inflammatory activation compared with control macrophages. At the whole animal level, hematopoietic cell-specific PFKFB3 disruption enhanced the effects of HFD feeding on promoting WAT inflammation, impairing WAT insulin signaling, and increasing systemic insulin resistance. In vitro, adipocytes co-cultured with PFKFB3-disrupted macrophages revealed increased proinflammatory responses and decreased insulin signaling compared with adipocytes co-cultured with control macrophages. These results suggest that PFKFB3 disruption in hematopoietic cells only exacerbates HFD-induced WAT inflammation and systemic insulin resistance.

Indole Alleviates Diet-Induced Hepatic Steatosis and Inflammation in a Manner Involving Myeloid Cell 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3.

BACKGROUND AND AIMS: Indole is a microbiota metabolite that exerts anti-inflammatory responses. However, the relevance of indole to human non-alcoholic fatty liver disease (NAFLD) is not clear. It also remains largely unknown whether and how indole acts to protect against NAFLD. The present study sought to examine the association between the circulating levels of indole and liver fat content in human subjects and explore the mechanisms underlying indole actions in mice with diet-induced NAFLD. APPROACH AND RESULTS: In a cohort of 137 subjects, the circulating levels of indole were reversely correlated with body mass index. In addition, the circulating levels of indole in obese subjects were significantly lower than those in lean subjects and were accompanied with increased liver fat content. At the whole-animal level, treatment of high-fat diet (HFD)-fed C57BL/6J mice with indole caused significant decreases in the severity of hepatic steatosis and inflammation. In cultured cells, indole treatment stimulated the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a master regulatory gene of glycolysis, and suppressed macrophage proinflammatory activation in a PFKFB3-dependent manner. Moreover, myeloid cell-specific PFKFB3 disruption exacerbated the severity of HFD-induced hepatic steatosis and inflammation and blunted the effect of indole on alleviating diet-induced NAFLD phenotype. CONCLUSIONS: Taken together, our results demonstrate that indole is relevant to human NAFLD and capable of alleviating diet-induced NAFLD phenotypes in mice in a myeloid cell PFKFB3-dependent manner. Therefore, indole mimetic and/or macrophage-specific PFKFB3 activation may be the viable preventive and/or therapeutic approaches for inflammation-associated diseases including NAFLD.

Adoptive transfer of Pfkfb3-disrupted hematopoietic cells to wild-type mice exacerbates diet-induced hepatic steatosis and inflammation.

BACKGROUND AND OBJECTIVES: Hepatic steatosis and inflammation are key characteristics of non-alcoholic fatty liver disease (NAFLD). However, whether and how hepatic steatosis and liver inflammation are differentially regulated remains to be elucidated. Considering that disruption of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3/iPfk2) dissociates fat deposition and inflammation, the present study examined a role for Pfkfb3/iPfk2 in hematopoietic cells in regulating hepatic steatosis and inflammation in mice. METHODS: Pfkfb3-disrupted (Pfkfb3 +/-) mice and wild-type (WT) littermates were fed a high-fat diet (HFD) and examined for NAFLD phenotype. Also, bone marrow cells isolated from Pfkfb3 +/- mice and WT mice were differentiated into macrophages for analysis of macrophage activation status and for bone marrow transplantation (BMT) to generate chimeric (WT/BMT- Pfkfb3 +/-) mice in which Pfkfb3 was disrupted only in hematopoietic cells and control chimeric (WT/BMT-WT) mice. The latter were also fed an HFD and examined for NAFLD phenotype. In vitro, hepatocytes were co-cultured with bone marrow-derived macrophages and examined for hepatocyte fat deposition and proinflammatory responses. RESULTS: After the feeding period, HFD-fed Pfkfb3 +/- mice displayed increased severity of liver inflammation in the absence of hepatic steatosis compared with HFD-fed WT mice. When inflammatory activation was analyzed, Pfkfb3 +/- macrophages revealed increased proinflammatory activation and decreased anti-proinflammatory activation. When NAFLD phenotype was analyzed in the chimeric mice, WT/BMT-Pfkfb3 +/- mice displayed increases in the severity of HFD-induced hepatic steatosis and inflammation compared with WT/BMT-WT mice. At the cellular level, hepatocytes co-cultured with Pfkfb3 +/- macrophages revealed increased fat deposition and proinflammatory responses compared with hepatocytes co-cultured with WT macrophages. CONCLUSIONS: Pfkfb3 disruption only in hematopoietic cells exacerbates HFD-induced hepatic steatosis and inflammation whereas the Pfkfb3/iPfk2 in nonhematopoietic cells appeared to be needed for HFD feeding to induce hepatic steatosis. As such, the Pfkfb3/iPfk2 plays a unique role in regulating NAFLD pathophysiology.

Mice lacking adenosine 2A receptor reveal increased severity of MCD-induced NASH.

Adenosine 2A receptor (A2AR) exerts a protective role in obesity-related non-alcoholic fatty liver disease. Here, we examined whether A2AR protects against non-alcoholic steatohepatitis (NASH). In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregation of macrophages in the liver compared with mice fed a chow diet. MCD feeding also significantly increased the numbers of A2AR-positive macrophages/Kupffer cells in liver sections although decreasing A2AR amount in liver lysates compared with chow diet feeding. Next, MCD-induced NASH phenotype was examined in A2AR-disrupted mice and control mice. Upon MCD feeding, A2AR-disruptd mice and control mice displayed comparable decreases in body weight and fat mass. However, MCD-fed A2AR-disrupted mice revealed greater liver weight and increased severity of hepatic steatosis compared with MCD-fed control mice. Moreover, A2AR-disupted mice displayed increased severity of MCD-induced liver inflammation, indicated by massive aggregation of macrophages and increased phosphorylation states of Jun-N terminal kinase (JNK) p46 and nuclear factor kappa B (NFκB) p65 and mRNA levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6. In vitro, incubation with MCD-mimicking media increased lipopolysaccharide (LPS)-induced phosphorylation states of JNK p46 and/or NFκB p65 and cytokine mRNAs in control macrophages and RAW264.7 cells, but not primary hepatocytes. Additionally, MCD-mimicking media significantly increased lipopolysaccharide-induced phosphorylation states of p38 and NFκB p65 in A2AR-deficient macrophages, but insignificantly decreased lipopolysaccharide-induced phosphorylation states of JNK p46 and NFκB p65 in A2AR-deficient hepatocytes. Collectively, these results suggest that A2AR disruption exacerbates MCD-induced NASH, which is attributable to, in large part, increased inflammatory responses in macrophages.

Regulation of adipose tissue inflammation by adenosine 2A receptor in obese mice.

Adenosine 2A receptor (A2AR) exerts anti-inflammatory effects. However, the role of A2AR in obesity-associated adipose tissue inflammation remains to be elucidated. The present study examined the expression of A2AR in adipose tissue of mice with diet-induced obesity and determined the effect of A2AR disruption on the status of obesity-associated adipose tissue inflammation. WT C57BL/6J mice and A2AR-disrupted mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and adipose tissue inflammation. In vitro, bone marrow-derived macrophages from A2AR-disrupted mice and WT control mice were treated with palmitate and examined for macrophage proinflammatory activation. Compared with that of low-fat diet (LFD)-fed WT mice, A2AR expression in adipose tissue of HFD-fed WT mice was increased significantly and was present predominantly in adipose tissue macrophages. The increase in adipose tissue A2AR expression in HFD-fed mice was accompanied with increased phosphorylation states of c-Jun N-terminal kinase 1 p46 and nuclear factor kappa B p65 and mRNA levels of interleukin (Il)-1beta, Il6 and tumor necrosis factor alpha. In A2AR-disrupted mice, HFD feeding induced significant increases in adipose tissue inflammation, indicated by enhanced proinflammatory signaling and increased proinflammatory cytokine expression, and adipose tissue insulin resistance, indicated by a decrease in insulin-stimulated Akt phosphorylation relative to those in WT mice. Lastly, A2AR disruption enhanced palmitate-induced macrophage proinflammatory activation. Taken together, these results suggest that A2AR plays a protective role in obesity-associated adipose tissue inflammation, which is attributable to, in large part, A2AR suppression of macrophage proinflammatory activation.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice.

BACKGROUND & AIMS: Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. METHODS: Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. RESULTS: Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. CONCLUSIONS: Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity.

Adenosine 2A receptor (A2A R) exerts protective roles in endotoxin- and/or ischemia-induced tissue damage. However, the role for A2A R in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell-specific A2A R disruption on the aspects of obesity-associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell-specific A2A R-disrupted mice and control mice were fed a high-fat diet (HFD) to induce NAFLD. In addition, bone marrow-derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A2A R-disrupted mice and myeloid cell-specific A2A R-defcient mice revealed increased severity of HFD-induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A2A R-deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild-type primary hepatocytes in macrophage-hepatocyte cocultures. In primary hepatocytes, A2A R deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A2A R deficiency significantly increased the abundance of sterol regulatory element-binding protein 1c (SREBP1c) in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A2A R, SREBP1c transcription activity was significantly increased in mouse hepatocytes. CONCLUSION: Taken together, our results demonstrate that disruption of A2A R in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. (Hepatology 2018;68:48-61).

Cyclic GMP-AMP Ameliorates Diet-induced Metabolic Dysregulation and Regulates Proinflammatory Responses Distinctly from STING Activation.

Endogenous cyclic GMP-AMP (cGAMP) binds and activates STING to induce type I interferons. However, whether cGAMP plays any roles in regulating metabolic homeostasis remains unknown. Here we show that exogenous cGAMP ameliorates obesity-associated metabolic dysregulation and uniquely alters proinflammatory responses. In obese mice, treatment with cGAMP significantly decreases diet-induced proinflammatory responses in liver and adipose tissues and ameliorates metabolic dysregulation. Strikingly, cGAMP exerts cell-type-specific anti-inflammatory effects on macrophages, hepatocytes, and adipocytes, which is distinct from the effect of STING activation by DMXAA on enhancing proinflammatory responses. While enhancing insulin-stimulated Akt phosphorylation in hepatocytes and adipocytes, cGAMP weakens the effects of glucagon on stimulating hepatocyte gluconeogenic enzyme expression and glucose output and blunts palmitate-induced hepatocyte fat deposition in an Akt-dependent manner. Taken together, these results suggest an essential role for cGAMP in linking innate immunity and metabolic homeostasis, indicating potential applications of cGAMP in treating obesity-associated inflammatory and metabolic diseases.

A role for PFKFB3/iPFK2 in metformin suppression of adipocyte inflammatory responses.

Metformin improves obesity-associated metabolic dysregulation, but has controversial effects on adipose tissue inflammation. The objective of the study is to examine the direct effect of metformin on adipocyte inflammatory responses and elucidate the underlying mechanisms. Adipocytes were differentiated from 3T3-L1 cells and treated with metformin at various doses and for different time periods. The treated cells were examined for the proinflammatory responses, as well as the phosphorylation states of AMPK and the expression of PFKFB3/iPFK2. In addition, PFKFB3/iPFK2-knockdown adipocytes were treated with metformin and examined for changes in the proinflammatory responses. The following results were obtained from the study. Treatment of adipocytes with metformin decreased the effects of lipopolysaccharide on inducing the phosphorylation states of JNK p46 and on increasing the mRNA levels of IL-1ß and TNFα. In addition, treatment with metformin increased the expression of PFKFB3/iPFK2, but failed to significantly alter the phosphorylation states of AMPK. In PFKFB3/iPFK2-knockdown adipocytes, treatment with metformin did not suppress the proinflammatory responses as did it in control adipocytes. In conclusion, metformin has a direct effect on suppressing adipocyte proinflammatory responses in an AMPK-independent manner. Also, metformin increases adipocyte expression of PFKFB3/iPFK2, which is involved in the anti-inflammatory effect of metformin.