We examined the effects of three high‐fat diets differing in the percentage of total calories from saturated fat (SF) (6%, 12%, and 24%), but identical in total fat (40%), on body composition, macrophage behavior, inflammation, and metabolic dysfunction in mice. Diets were administered for 16 weeks. Body composition and metabolism (glucose, insulin, triglycerides, LDL‐C, HDL‐C, total cholesterol) were examined monthly. Adipose tissue (AT) expression of marker genes for M1 and M2 macrophages and inflammatory mediators (TLR‐2, TLR‐4, MCP‐1, TNF‐α, IL‐6, IL‐10, SOCS1, IFN‐γ) was measured along with activation of NFκB, JNK and p38‐MAPK. AT macrophage infiltration was examined using immunohistochemistry. Circulating MCP‐1, IL‐6, adiponectin, and leptin were also measured. SF content, independent of total fat, can profoundly affect adiposity, macrophage behavior, inflammation, and metabolic dysfunction. In general, the 12%‐SF diet, most closely mimicking the standard American diet, led to the greatest adiposity, macrophage infiltration, and insulin resistance (IR), whereas the 6%‐SF and 24%‐SF diets produced lower levels of these variables with the 24%‐SF diet resulting in the least degree of IR and the highest TC/HDL‐C ratio. Macrophage behavior, inflammation and IR following HFDs are heavily influenced by dietary SF content, however, these responses are not necessarily proportional to the SF%. This work was supported by an ASPIRE (Advanced Support Programs for Innovative Research Excellence) grant from the University of South Carolina to E.A.M. Grant Funding Source: ASPIRE

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc Min/+, Apc Min/+/MCP-1−/− or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, β-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% ( P < 0.05). This was consistent with an increase in apoptotic cells ( P < 0.05), but there was no change detected in proliferation or β-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue ( P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-β, and CCL17) ( P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells ( P < 0.05). In addition, MCP-1−/− offset the increased mRNA expression of IL-1β and IL-6 in intestinal tissue and IL-1β and TNF-α in polyp tissue ( P < 0.05), and prevented the decrease in SOCS1 expression ( P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

Obesity is characterized by chronic low-grade, systemic inflammation, altered gut microbiota, and gut barrier disruption. Additionally, obesity is associated with increased activity of endocannabinoid system (eCB). However, the clear connection between gut microbiota and the eCB system in the regulation of energy homeostasis and adipose tissue inflammation and metabolism, remains to be established. We investigated the effect of treatment of mice with a cannabinoid receptor 1 (CB1) antagonist on Diet-Induced Obesity (DIO), specifically whether such a treatment that blocks endocannabinoid activity can induce changes in gut microbiota and anti-inflammatory state in adipose tissue. Blockade of CB1 attenuated DIO, inflammatory cytokines and trafficking of M1 macrophages into adipose tissue. Decreased inflammatory tone was associated with a lower intestinal permeability and decreased metabolic endotoxemia as evidenced by reduced plasma LPS level, and improved hyperglycemia and insulin resistance. 16S rRNA metagenomics sequencing revealed that CB1 blockade dramatically increased relative abundance of Akkermansia muciniphila and decreased Lanchnospiraceae and Erysipelotrichaceae in the gut. Together, the current study suggests that blocking of CB1 ameliorates Diet-Induced Obesity and metabolic disorder by modulating macrophage inflammatory mediators, and that this effect is associated with alterations in gut microbiota and their metabolites.

SummaryInflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses.

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) has become an epidemic largely due to the worldwide increase in obesity. While lifestyle modifications and pharmacotherapies have been used to alleviate NAFLD, successful treatment options are limited. One of the main barriers to finding safe and effective drugs for long-term use in NAFLD is the fast initiation and progression of disease in the available preclinical models. Therefore, we are in need of preclinical models that (1) mimic the human manifestation of NAFLD and (2) have a longer progression time to allow for the design of superior treatments. AIM To characterize a model of prolonged high-fat diet (HFD) feeding for investigation of the long-term progression of NAFLD. METHODS In this study, we utilized prolonged HFD feeding to examine NAFLD features in C57BL/6 male mice. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 80 wk to promote obesity (Old-HFD group, n = 18). A low-fat diet (LFD) (14% fat, 32% protein, and 54% carbohydrate) was administered for the same duration to age-matched mice (Old-LFD group, n = 15). An additional group of mice was maintained on the LFD (Young-LFD, n = 20) for a shorter duration (6 wk) to distinguish between age-dependent and age-independent effects. Liver, colon, adipose tissue, and feces were collected for histological and molecular assessments. RESULTS Prolonged HFD feeding led to obesity and insulin resistance. Histological analysis in the liver of HFD mice demonstrated steatosis, cell injury, portal and lobular inflammation and fibrosis. In addition, molecular analysis for markers of endoplasmic reticulum stress established that the liver tissue of HFD mice have increased phosphorylated Jnk and CHOP. Lastly, we evaluated the gut microbial composition of Old-LFD and Old-HFD. We observed that prolonged HFD feeding in mice increased the relative abundance of the Firmicutes phylum. At the genus level, we observed a significant increase in the abundance of Adercreutzia, Coprococcus, Dorea , and Ruminococcus and decreased relative abundance of Turicibacter and Anaeroplasma in HFD mice. CONCLUSION Overall, these data suggest that chronic HFD consumption in mice can mimic pathophysiological and some microbial events observed in NAFLD patients.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice ( n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle ( week 7) and twice weekly until euthanasia, mice ( n = 16/group) received either 200 μl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% ( P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFβ, CCL17) in the colon tissue ( P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum ( P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

Many observational epidemiologic studies suggest an association between high-fat-diet (HFD) and colon cancer risk. However, the lack of controlled experimental studies that examine this relationship and the mechanisms involved weaken the basis for inferring a causal relationship. Inflammation plays a role in colon cancer progression and HFDs have been reported to increase inflammation; however, the inflammatory effects of HFD in colon cancer have yet to be firmly established. We examined the effects of a novel HFD that closely mimics the standard American diet (12% and 40% of total caloric intake from saturated fat and total fat, respectively) on macrophage markers and inflammatory mediators in a mouse model of intestinal tumorigenesis and relate this to polyp characteristics as well as measures of adiposity. Male ApcMin/+ mice (7–8/group) were fed a Control Diet (Con) or novel high-fat-diet (HFD) from 4 to 12 weeks of age. Body weight and body composition were measured weekly and monthly, respectively. Intestinal tissue was analyzed for polyp burden (number and size). Gene expression of macrophage markers and inflammatory mediators were examined in the adipose tissue and polyps. The HFD increased the expression of macrophage markers and inflammatory mediators in the adipose tissue (F4/80, CD11c, TLR-4 and MCP-1) and tumor microenvironment (IL-12, MCP-1, IL-6 and TNF-α). As expected, the HFD increased body weight, body fat percent, fat mass and blood glucose (P < 0.05), and was associated with an increase in the number of large polyps (P < 0.05) but not total polyps. In summary, consumption of a HFD, similar in macronutrient composition to the standard American diet, altered the expression of macrophage phenotypic markers and inflammatory mediators in adipose tissue and intestinal polyps and this was associated with increased tumorigenesis.

Cancer cachexia is a muscle wasting condition that occurs in response to a malignant growth in the body. The mechanisms regulating cardiac muscle mass with cachexia are not well understood. Using the ApcMin/+ mouse model of colorectal cancer, we investigated how cachexia affects the regulation of 5′-adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling in the heart. Compared to age-matched C57BL/6 (BL6) mice, ApcMin/+ body mass and heart mass were lower at 12 (11±5 and 8±3%, respectively) and 20 weeks (26±3 and 6±4%, respectively) of age (P<0.05). Diminished heart mass in the 20-week-old ApcMin/+ mice coincided with a decreased rate of myofibrillar protein synthesis and increased AMPKα phosphorylation. Cachexia decreased mTOR phosphorylation and the phosphorylation of the mTOR substrates, S6 ribosomal protein and 4EBP1 independent of Akt activation. These changes in mTOR-related protein signaling were accompanied by modest increases in the amount of Beclin1 but not protein ubiquitination or cardiomyocyte apoptosis. Taken together, these data suggest that loss of cardiac mass during cachexia progression in the ApcMin/+ mouse is associated with an Akt-independent suppression of anabolic signaling and evidence of increased autophagy.