Emodin reduces tumor burden by diminishing M2-like macrophages in colorectal cancer

Emodin, a natural anthraquinone, has been shown to have anti-tumorigenic properties and may be an effective therapy for colorectal cancer (CRC). However, its clinical development has been hampered by a poor understanding of its mechanism of action. The purpose of this study was to 1) evaluate the efficacy of emodin in mouse models of intestinal/colorectal cancer and 2) to examine the impact of emodin on macrophage behavior in the context of CRC. We utilized a genetic model of intestinal cancer (ApcMin/+) and a chemically induced model of CRC (AOM/DSS). Emodin was administered orally (40 mg/kg or 80 mg/kg in AOM/DSS and 80mg/kg in ApcMin/+) 3x/week to observe its preventative effects. Emodin reduced polyp count and size in both rodent models (p<0.05). We further analyzed the colon microenvironment of AOM/DSS mice and found that mice treated with emodin exhibited lower pro-tumorigenic M2-like macrophages and a reduced ratio of M2/M1 macrophages within the colon (p<0.05). Despite this, we did not detect any significant changes in M2-associated cytokines (IL10, IL4, and Tgfb1) nor M1-associated cytokines (IL6, TNFα, IL1β, and IFNγ) within excised polyps. However, there was a significant increase in NOS2 expression (M1 marker) in mice treated with 80 mg/kg emodin (p<0.05). To confirm emodin's effects on macrophages, we exposed bone marrow-derived macrophages (BMDMs) to C26 colon cancer cell conditioned media. Supporting our in vivo data, emodin reduced M2-like macrophages. Overall, these data support the development of emodin as a natural compound for prevention of CRC given its ability to target pro-tumor macrophages.

MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells

Background: In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. Methods: MAPK p38, Ulk1 phosphorylation and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. Results: We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1β induction, which depended on the MAPK p38/Ulk1 pathway. Conclusions: Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1/Atg13 disruption, which inhibits autophagy and induces IL-1β expression in HSCs.

Exposure to binge ethanol and fatty acid ethyl esters exacerbates chronic ethanol-induced pancreatic injury in hepatic alcohol dehydrogenase deficient deer mice

Alcoholic chronic pancreatitis (ACP) is a fibroinflammatory disease of the pancreas. However, metabolic basis of ACP is not clearly understood. In this study, we evaluated differential pancreatic injury in hepatic alcohol dehydrogenase deficient (ADH-) deer mice fed chronic ethanol (EtOH), chronic plus binge EtOH, and chronic plus binge EtOH and fatty acid ethyl esters (FAEEs, nonoxidative metabolites of EtOH) to understand the metabolic basis of ACP. Hepatic ADH- and ADH normal (ADH+) deer mice were fed Lieber-DeCarli liquid diet containing 3% (w/v) EtOH for three months. One week before the euthanization, chronic EtOH fed mice were further administered with an oral gavage of binge EtOH with/without FAEEs. Blood alcohol concentration (BAC), pancreatic injury and inflammatory markers were measured. Pancreatic morphology, ultrastructural changes, endoplasmic reticulum (ER)/oxidative stress were examined using H & E staining, electron microscopy, immunostaining, and/or Western blot, respectively. Overall, BAC was substantially increased in chronic EtOH fed groups of ADH- vs. ADH+ deer mice. A significant change in pancreatic acinar cell morphology, with mild to moderate fibrosis and ultrastructural changes evident by dilatations and disruption of ER cisternae, ER/oxidative stress along with increased levels of inflammatory markers were observed in the pancreas of chronic EtOH fed groups of ADH- vs. ADH+ deer mice. Furthermore, chronic plus binge EtOH and FAEEs exposure elevated BAC, enhanced ER/oxidative stress and exacerbated chronic EtOH-induced pancreatic injury in ADH- deer mice suggesting a role of increased body burden of EtOH and its metabolism under reduced hepatic ADH in initiation and progression of ACP.

The characteristics of mesenteric adipose tissue attached to different intestinal segments and their roles in immune regulation

Background: Mesenteric adipose tissue (MAT) plays a critical role in the intestinal physiological ecosystems. Small and large intestines have evidently intrinsic and distinct characteristics. However, whether there exist any mesenteric differences adjacent to the small and large intestines (SMAT and LMAT) has not been properly characterized. We studied the important facets of these differences, such as morphology, gene expression, cell components and immune regulation of MATs, to characterize the mesenteric differences. Methods: The SMAT and LMAT of mice were utilized for comparison of tissue morphology. Paired mesenteric samples were analyzed by RNA-seq to clarify gene expression profiles. MAT partial excision models were constructed to illustrate the immune regulation roles of MATs, and 16S-seq was applied to detect the subsequent effect on microbiota. Results: Our data show that different segments of mesenteries have different morphological structures. SMAT not only has smaller adipocytes but also contains more fat-associated lymphoid clusters than LMAT. The gene expression profile is also discrepant between these two MATs in mice. B-cell markers were abundantly expressed in SMAT, while development-related genes were highly expressed in LMAT. Adipose-derived stem cells of LMAT exhibited higher adipogenic potential and lower proliferation rates than those of SMAT. In addition, SMAT and LMAT play different roles in immune regulation and subsequently affect microbiota components. Finally, our data clarified the described differences between SMAT and LMAT in humans. Conclusions: There were significant differences in cell morphology, gene expression profiles, cell components, biological characteristics, and immune and microbiota regulation roles between regional MATs.

Maternal Obesogenic Diet Regulates Offspring Bile Acid Homeostasis and Hepatic Lipid Metabolism via the Gut Microbiome in Mice

Mice exposed in gestation to maternal high fat/high sucrose (HF/HS) diet develop altered bile acid (BA) homeostasis. We hypothesized that these reflect an altered microbiome and asked if microbiota transplanted from HF/HS offspring change hepatic BA and lipid metabolism to determine the directionality of effect. Female mice were fed HF/HS or chow (CON) for 6 weeks and bred with lean males. 16S sequencing was performed to compare taxa in offspring. Cecal microbiome transplantation (CMT) was performed from HF/HS or CON offspring into antibiotic treated mice fed chow or high fructose. BA, lipid metabolic, and gene expression analyses performed in recipient mice. Gut microbiomes from HF/HS offspring segregated from CON offspring, with increased Firmicutes to Bacteriodetes ratios and Verrucomicrobial abundance. Following CMT, HF/HS recipient mice had larger BA pools, and increased intrahepatic muricholic acid and decreased deoxycholic acid species. HF/HS recipient mice exhibited downregulated hepatic Mrp2, increased hepatic Oatp1b2, and decreased ileal Asbt mRNA expression. HF/HS recipient mice exhibited decreased cecal butyrate and increased hepatic expression of Il6. HF/HS recipient mice had larger livers, and increased intrahepatic triglyceride versus CON recipient mice after fructose feeding, with increased hepatic mRNA expression of lipogenic genes including Srebf1, Fabp1, Mogat1, and Mogat2. CMT from HF/HS offspring increased BA pool and shifted the composition of the intrahepatic BA pool. CMT from HF/HS donor offspring increased fructose-induced liver triglyceride accumulation. These findings support a causal role for vertical transfer of an altered microbiome in hepatic BA and lipid metabolism in HF/HS offspring.

Developmental Alterations of Intestinal SGLT1 and GLUT2 Induced by Early Weaning Coincides with Persistent Low-Grade Metabolic Inflammation in Female Pigs

Early life adversity (ELA) is linked with the increased risk for inflammatory and metabolic diseases in later life but the mechanisms remain poorly understood. Intestinal epithelial glucose transporters SGLT1 and GLUT2 are the major route for intestinal glucose uptake but have also received increased attention as modulators of inflammatory and metabolic diseases. Here we tested the hypothesis that early weaning (EW) in pigs, an established model of ELA, alters the development of epithelial glucose transporters and coincides with elevated markers of metabolic inflammation. Jejunum and ileum of 90 d old pigs previously exposed to EW (16 d wean age), exhibited reduced SGLT1 activity (by ~ 30%, P<0.05), compared with late weaned (LW, 26 d wean age) controls . In contrast, GLUT2-mediated glucose transport was increased (P = 0.003) in EW pigs compared with LW pigs. Reciprocal changes in SGLT1 and GLUT2-mediated transport coincided with transporter protein expression in the intestinal brush border membranes (BBM) that were observed at 90 d and 150 d of age. Ileal SGLT1-mediated glucose transport and BBM expression were Inhibited by the β-adrenergic receptor (βAR) blocker propranolol in EW and LW pigs. In contrast, propranolol enhanced ileal GLUT2-mediated glucose transport (P=0.015) and BBMV abundance (P=0.035) LW pigs, but not EW pigs. Early weaned pigs exhibited chronic elevated blood glucose and C-Reactive Protein (CRP) levels, and adipocyte hypertrophy and upregulated adipogenesis-related gene expression in visceral adipose tissue. Altered development of intestinal glucose transporters by EW could underlie the increased risk for later life inflammatory and metabolic diseases.

Stiffness is associated with hepatic stellate cell heterogeneity during liver fibrosis

The fibrogenic wound-healing response in liver increases stiffness. Stiffness mechano-transduction in turn amplifies fibrogenesis. Here, we aimed to understand the distribution of stiffness in fibrotic liver, how it impacts hepatic stellate cell (HSC) heterogeneity and identify mechanisms by which stiffness amplifies fibrogenic responses. Magnetic resonance elastography and atomic force microscopy demonstrated a heterogenous distribution of liver stiffness at macroscopic and microscopic levels, respectively, in a carbon tetrachloride (CCl4) mouse model of liver fibrosis as compared to controls. High stiffness was mainly attributed to extracellular matrix dense areas. To identify a stiffness-sensitive HSC sub-population, we performed scRNA-seq on primary HSCs derived from healthy versus CCl4-treated mice. A sub-cluster of HSCs was matrix-associated with the most upregulated pathway in this sub-population being focal adhesion signaling, including a specific protein termed four and a half LIM domains protein 2 (FHL2). In vitro, FHL2 expression was increased in primary human HSCs cultured on stiff matrix as compared to HSCs on soft matrix. Moreover, FHL2 knockdown inhibited fibronectin and collagen 1 expression, whereas its overexpression promoted matrix production. In summary, we demonstrate stiffness heterogeneity at the whole organ, lobular, and cellular level which drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Xenobiotic receptors and the regulation of intestinal homeostasis - harnessing the chemical output of the intestinal microbiota

The commensal bacteria that reside in the gastrointestinal tract exist in a symbiotic relationship with the host, driving the development of the immune system and maintaining metabolic and tissue homeostasis in the local environment. The intestinal microbiota has the capacity to generate a wide array of chemical metabolites to which the cells of the intestinal mucosa are exposed. Host cells express xenobiotic receptors, such as the aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), that can sense and respond to chemicals that are generated by non-host pathways. In this review, we will outline the physiological and immunological processes within the intestinal environment that are regulated by microbial metabolites through the activation of the AhR and PXR, with a focus on ligands generated by the step-wise catabolism of tryptophan.

LPS induces rapid increase in GDF15 levels in mice, rats and humans but is not required for anorexia in mice

Growth differentiation factor 15 (GDF15), a TGFβ superfamily cytokine, acts through its receptor, GDNF-family receptor α-like (GFRAL), to suppress food intake and promote nausea. GDF15 is broadly expressed at low levels but increases in states of disease such as cancer, cachexia, and sepsis. Whether GDF15 is necessary for inducing sepsis associated anorexia and body weight loss is currently unclear. To test this we used a model of moderate systemic infection in GDF15KO and GFRALKO mice with lipopolysaccharide (LPS) treatment to define the role of GDF15 signaling in infection-mediated physiologic responses. Since physiologic responses to LPS depend on housing temperature, we tested the effects of subthermoneutral and thermoneutral conditions on eliciting anorexia and inducing GDF15. Our data demonstrate a conserved LPS-mediated increase in circulating GDF15 levels in mouse, rat and human. However, we did not detect differences in LPS induced anorexia between WT and GDF15KO or GFRALKO mice. Further, there were no differences in anorexia or circulating GDF15 levels at either thermoneutral or subthermoneutral housing conditions in LPS treated mice. These data demonstrate that GDF15 is not necessary to drive food intake suppression in response to moderate doses of LPS.

Hyperuricemia induces lipid disturbances by upregulating the CXCL-13 pathway

The molecular mechanism underlying hyperuricemia-induced lipid metabolism disorders is not clear. The purpose of the current study was to investigate the mechanism of lipid disturbances in a hyperuricemia mice model. RNAseq showed that differentially expressed genes (DEGs) in the fatty acid synthesis signaling pathway were mainly enriched, and CXCL-13 was significantly enriched in protein-protein interaction networks. Western blotting, Q-PCR, and immunofluorescence results further showed that hyperuricemia upregulated CXCL-13 and disturbed lipid metabolism in vivo and in vitro. Furthermore, CXCL-13 alone also promoted the accumulation of lipid droplets and upregulated the expression of FAS and SREBP1, blocking AMPK signaling and activating the PKC and P38 signaling pathways. Silencing CXCL-13 reversed uric-acid-induced lipid droplet accumulation, which further downregulated FAS and SREBP1 expression, inhibited the p38 and PKC signaling, and activated AMPK signaling. In conclusion, hyperuricemia induces lipid metabolism disorders via the CXCL-13 pathway, making CXCL-13 a key regulatory factor linking hyperuricemia and lipid metabolism disorders. These results may provide novel insights for the treatment of hyperuricemia.