Differential Effects of Short-Chain Fatty Acids on L6 Myotube Inflammatory Mediator Production in Response to Lipopolysaccharide- or Palmitic Acid-Stimulation.

Short-chain fatty acids (SCFA) produced from dietary non-digestible carbohydrate fermentation have metabolic effects in skeletal muscle; however, their effect on inflammatory mediator production is unknown. In this study, L6 myotubes were cultured with individual SCFA (acetate, propionate, and butyrate) at 0.5 mM and 2.5 mM ± 10 ng/mL lipopolysaccharide (LPS) or ± 500 µM palmitic acid (PA) for 24 h. In response to LPS, only butyrate had an effect at the lower concentration (0.5 mM), whereas at the higher concentration (2.5 mM) both propionate and butyrate reduced MCP-1, MIP-1α, and RANTES secretion (p < 0.05), and only butyrate reduced IL-6 secretion and intracellular protein levels of phospho-STAT3 (p < 0.05). In response to PA, 0.5 mM butyrate reduced protein expression of phospho-NFκB p65 and the secretion of IL-6, MIP-1α, and MCP-1, whereas all three SCFA reduced RANTES secretion (p < 0.05). At the 2.5 mM SCFA concentration combined with PA stimulation, all three SCFA reduced intracellular protein expression of phospho-NFκB p65 and phospho-STAT3 and secreted protein levels of MCP-1, IL-6, and RANTES, whereas only butyrate reduced secretion of MIP-1α (p < 0.05). Thus, SCFA exhibit differential effects on inflammatory mediator expression in response to LPS and PA stimulation, which has implications for their individual impacts on inflammation-mediated skeletal muscle dysfunction.

Dietary ω-3 polyunsaturated fatty acids modulate CD4+ T-cell subset markers, adipocyte antigen-presentation potential, and NLRP3 inflammasome activity in a coculture model of obese adipose tissue.

OBJECTIVES: Chronic low-grade inflammation in obesity is partly driven by inflammatory cross talk between adipocytes and interferon-γ-secreting CD4+ T-helper (Th)1 cells, a process we have shown may be mitigated by long-chain (LC) ω-3 polyunsaturated fatty acids (PUFAs). Our objective was to study pivotal mediators of interactions between Th1 cells and adipocytes as potential mechanisms underlying the antiinflammatory effects of LC ω-3 PUFAs. METHODS: Using an in vitro model, 3T3-L1 adipocytes were cocultured with purified splenic CD4+ T cells from C57BL/6 mice consuming one of two isocaloric high-fat (HF) diets (60% kcal fat), containing either 41.2% kcal from lard + 18.7% kcal from corn oil (control, HF) or 41.2% kcal from lard + 13.4% kcal from corn oil + 5.3% kcal from fish oil (HF+FO). Cocultures were stimulated for 48 h with lipopolysaccharide (10 ng/mL). RESULTS: Compared with HF cocultures, HF+FO reduced Th1-cell markers (including secreted interferon-γ) and increased Th2-cell markers, consistent with reduced expression of genes related to major histocompatibility complex II (P < 0.05). HF+FO also blunted markers of priming and activity of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (P < 0.05). In confirmatory work, 3T3-L1 adipocyte pretreatment with the LC ω-3 PUFA docosahexaenoic acid (100 µM, 24 h) blunted interferon-γ-induced (5 ng/mL, 24 h) expression of genes related to major histocompatibility complex II and priming and activity markers of the NLRP3 inflammasome compared with control (P < 0.05). CONCLUSIONS: Inflammatory interactions between CD4+ T cells and adipocytes may provide a target for LC ω-3 PUFAs to mitigate obesity-associated inflammation.

Fish oil supplementation increases expression of mammary tumor apoptosis mediators and reduces inflammation in an obesity-associated HER-2 breast cancer model.

Obesity is associated with inflammation and has been shown to increase breast cancer severity. The objective of this study was to examine the effect of fish oil (FO) supplementation in obesity-associated mammary tumorigenesis in the MMTV-neu(ndl)-YD5 mouse model of human epidermal growth factor receptor-2 positive BC. Female mice were fed one of three diets for 16 weeks: i) high fat diet [HF, % kacl: 41.2% lard, 18.7% corn oil (CO)], ii) an isocaloric HF plus menhaden FO diet (HF+FO, % kcal: 41.2 lard, 13.4% CO, 5.3% FO), iii) low fat diet (LF, % kcal: 4.7% lard, 6% CO). HF mice had increased body weight, visceral adipose weight and serum hormone concentrations (increased leptin and resistin; decreased adiponectin) versus LF, which was attenuated in the HF+FO group versus HF (P<.05). Compared to HF, tumor onset was delayed in HF+FO and LF mice (P<0.05). Compared to HF, HF+FO reduced mammary tumor multiplicity (-27%), tumor weight (-46%) and total tumor volume (-50%) (P<0.05). Additionally, HF+FO reduced mammary tumor multiplicity (-33%), tumor weight (-39%) and total tumor volume (-60%) versus LF. HF+FO improved mammary tumor apoptosis status with increased expression of pro-apoptotic Bad and decreased expression of anti-apoptotic Bcl-xLmediators versus HF (P<0.05). Additionally, HF+FO decreased tumor protein expression of activated Akt, NFκB p65 and STAT3, versus HF (P<0.05). Tumor mRNA expression of inflammatory mediators TNFα, IL-6 and leptin were reduced in HF+FO, whereas IL-10 expression was increased compared to HF (P<0.05). Collectively these results demonstrate the efficacy of FO supplementation for improving obesity-associated breast cancer outcomes.

Studying Adipocyte and Immune Cell Cross Talk Using a Co-culture System.

The co-culture of adipocytes and immune cells, such as macrophages or T cells (CD4+ or CD8+ subsets), is a novel experimental approach used to study paracrine interactions (or the cross talk) between cultured cell types in isolation, in order to understand their role in obese adipose tissue (AT) inflammation and dysfunction. Here we describe the general methodologies required for the co-culture of mature adipocytes (differentiated 3T3-L1 pre-adipocyte cell line) with primary immune cell subsets purified from mouse splenic mononuclear cells using a magnetic MicroBead positive selection, wherein multiple immune cell populations can be purified sequentially from a single mouse spleen, thereby providing diversity in the types of immune cells that can be co-cultured with adipocytes. Additionally, we describe experimental procedures for co-culturing adipocytes and immune cells in two different co-culture systems, including a cell contact-dependent co-culture system, wherein the cells are in direct physical contact, and a cell contact-independent, soluble mediator-driven co-culture system wherein the cells are physically separated by a trans-well semipermeable membrane. Finally, we discuss how these co-culture models can be utilized to recapitulate the AT microenvironment in obesity by utilizing physiologically relevant ratios of adipocytes:immune cells (specifically CDllb+ macrophages, CD4+ T cells, or CD8+ T cells) and lipopolysaccharide stimulation that mimics endotoxin concentrations observed in obesity.

Dietary long-chain n-3 PUFAs mitigate CD4+ T cell/adipocyte inflammatory interactions in co-culture models of obese adipose tissue.

Obese adipose tissue (AT) inflammation is partly driven by accumulation of CD4+ T helper (Th)1 cells and reduced Th2 and T regulatory subsets, which promotes macrophage chemotaxis and ensuing AT metabolic dysfunction. This study investigated CD4+ T cell/adipocyte cytokine-mediated paracrine interactions (cross talk) as a target for dietary intervention to mitigate obese AT inflammation. Using an in vitro co-culture model designed to recapitulate CD4+ T cell accumulation in obese AT (5% of stromal vascular cellular fraction), 3T3-L1 adipocytes were co-cultured with purified splenic CD4+ T cells from C57Bl/6 mice consuming one of two isocaloric diets containing either 10% w/w safflower oil (control, CON) or 7% w/w safflower oil+3% w/w fish oil (FO) for 4 weeks (n=8-11/diet). The FO diet provided 1.9% kcal from the long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid, a dose that can be achieved by supplementation. Co-cultures were stimulated for 48 h with lipopolysaccharide (LPS) to mimic in vivo obese endotoxin levels or with conditioned media collected from LPS-stimulated visceral AT isolated from CON-fed mice. In both stimulation conditions, FO reduced mRNA expression and/or secreted protein levels of Th1 markers (T-bet, IFN-γ) and increased Th2 markers (GATA3, IL-4), concomitant with reduced inflammatory cytokines (IL-1ß, IL-6, IL-12p70, TNF-α), macrophage chemokines (MCP-1, MCP-3, MIP-1α, MIP-2) and levels of activated central regulators of inflammatory signaling (NF-κB, STAT-1, STAT-3) (P<.05). Therefore, CD4+ T cell/adipocyte cross talk represents a potential target for LC n-3 PUFAs to mitigate obese AT inflammation.

CD8+ T cell/adipocyte inflammatory cross talk and ensuing M1 macrophage polarization are reduced by fish-oil-derived n-3 polyunsaturated fatty acids, in part by a TNF-α-dependent mechanism.

Obese visceral adipose tissue (AT) inflammation is driven by adipokine-mediated cross talk between CD8+ T cells and adipocytes, a process mitigated by long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) but underlying mechanisms and ensuing effects on macrophage polarization status are unknown. Using an in vitro co-culture model that recapitulates the degree of CD8+ T cell infiltration reported in obese AT, 3T3-L1 adipocytes were co-cultured for 24 h with purified splenic CD8+ T cells from C57Bl/6 mice consuming either a 10% w/w safflower oil (control, CON) or 7% w/w safflower oil + 3% w/w fish oil (FO) diet for 4 weeks (n=8-10/diet). Co-cultured cells were in direct contact or in a contact-independent condition separated by a Transwell permeable membrane and stimulated with lipopolysaccharide (10 ng/ml) to mimic in vivo obese endotoxin levels. In contact-dependent co-cultures, FO reduced inflammatory (IL-6, TNFα, IFN-γ) and macrophage chemotactic (CCL2, CCL7, CCL3) mRNA expression and/or secreted protein, NF-κB p65 activation, ROS accumulation, NLRP3 inflammasome priming (Nlrp3, Il1ß mRNA) and activation (caspase-1 activity) compared to CON (P<.05). The anti-inflammatory action of FO was reproduced by the addition of a TNF-α neutralizing antibody (1 µg/ml) to CON co-cultures (CON/anti-TNF-α), albeit to a lesser degree. Conditioned media from FO and CON/anti-TNF-α co-cultures, in turn, reduced RAW 264.7 macrophage mRNA expression of M1 polarization markers (iNos, Cd11c, Ccr2) and associated inflammatory cytokines (Il6, Tnfα, Il1ß) compared to CON. These data suggest that inflammatory CD8+ T cell/adipocyte cross talk is partially attributable to TNF-α signaling, which can be mitigated by LC n-3 PUFA.

Fish oil supplementation to a high-fat diet improves both intestinal health and the systemic obese phenotype.

Impaired intestinal health characterized by a dysbiotic microbial community and a dysfunctional epithelial barrier contributes to host inflammation and metabolic dysfunction in obesity. Fish oil (FO)-derived n-3 polyunsaturated fatty acids have been shown to improve aspects of the obese phenotype; however, their effect on obese intestinal health is unknown. This study aimed to determine the effect of dietary FO on the intestinal microenvironment, including the microbial community and epithelial barrier, in a mouse model of high-fat diet induced obesity and metabolic dysfunction. Male C57BL/6 mice were fed (12 weeks) either a high-fat diet (HF, 60% fat as kcal) or an isocaloric HF supplemented with Menhaden FO (5.3% kcal, HF + FO). 16S rRNA sequencing was used to determine changes in fecal microbiota. Intestinal (ileum and colon) and epididymal adipose tissue RNA was used to assess biomarkers of barrier integrity and inflammatory status, respectively. Serum was used to assess adipokine concentrations and insulin resistance. HF + FO diet altered the fecal microbiota by decreasing the abundance of Firmicutes and increasing the abundance of members of the Bacteroidetes phyla, as well as increasing the abundance of antiobesogenic Akkermansia muciniphila, compared to HF. Intestinal epithelial barrier functions were improved by HF + FO evidenced by increased mRNA expression of tight junction components, antimicrobial defenses and mucus barrier components. HF + FO-fed mice exhibited improvements in homeostatic model assessment of insulin resistance, oral glucose tolerance and serum adipokine concentrations and epididymal mRNA expression (increased adiponectin and decreased leptin) versus HF. HF + FO improved obese intestinal health and attenuated metabolic dysfunction associated with obesity.

Navy bean supplemented high-fat diet improves intestinal health, epithelial barrier integrity and critical aspects of the obese inflammatory phenotype.

Obesity is associated with impaired intestinal epithelial barrier function and an altered microbiota community structure, which contribute to host systemic inflammation and metabolic dysfunction. Fiber-rich common beans (Phaseolus vulgaris) promote intestinal health (microbiota and host epithelial barrier integrity) in lean mice. The objective was to assess the intestinal health promoting effects of navy bean supplementation during high-fat (HF)diet-induced obesity. Male C57BL/6 mice were fed either a high-fat (HF) diet (60% of kcal from fat) or an isocaloric HF diet supplemented with 15.7% (by weight) cooked navy bean powder (HF+B) for 12 weeks. Compared to HF, the HF+B diet altered the fecal microbiota community structure (16S rRNA gene sequencing), most notably increasing abundance of Akkermansia muciniphila (+19-fold), whose abundance typically decreases in obese humans and rodents. Additionally, HF+B fecal abundance of carbohydrate fermenting, short chain fatty acid (SCFA) producing Prevotella (+332-fold) and S24-7 (+1.6-fold) and fecal SCFA levels were increased. HF+B improved intestinal health and epithelial barrier integrity versus HF, evidenced by reduced serum fluorescein isothiocyanate (FITC)-dextran concentration in an in vivo gut permeability test, and increased intestinal mRNA expression of tight junction components (ZO-1, occludin), anti-microbial defenses (Reg3γ, IgA, Defα5, Defß2) and mucins (Muc2). Additionally, HF+B improved the systemic obese phenotype via reduced serum HOMA-IR and leptin:adiponectin ratio, and locally via attenuation of epididymal adipose tissue crown-like structure formation, adipocyte size, and inflammatory transcription factor (NFκBp65 and STAT3) activation. Therefore, navy bean supplementation improved obese intestinal health (microbiota and epithelial barrier integrity) and attenuated the severity of the obese phenotype.

Navy and black bean supplementation attenuates colitis-associated inflammation and colonic epithelial damage.

The enriched levels of nondigestible fermentable carbohydrates and phenolic compounds found in common beans can exert immunomodulatory effects within the colon that improve gut health and mitigate the severity of colitis-associated inflammatory pathology. Prior to acute colitis onset, C57Bl/6 mice were prefed isocaloric 20% cooked navy bean (NB) or black bean (BB) diets for 3 weeks and switched to control basal diet (BD) 24 h prior to colitis induction via 5-day exposure to dextran sodium sulfate (2% w/v in drinking water)+3 days of fresh water. The severity of the acute colitis phenotype was attenuated by bean prefeeding, evidenced by reduced colon tissue inflammatory transcription factor activation (NFκB, STAT3) and inflammatory mediator levels in the colon (IL-1ß, IL-6, IL-18 and MCP-1) and serum (TNFα, IL-6, IL-1ß, MCP-1) versus BD (P≤.05). Additionally, biomarkers of enhanced wound repair responses were increased by bean prefeeding including colon tissue protein levels of IL-22, IL-27 and activated (i.e., GTP-bound) Cdc42 and Rac1 versus BD (P≤.05). mRNA expressions of genes involved in normal colonic epithelial function and the promotion of epithelial barrier integrity, defense and/or restitution and wound closure including MUC1, RELMß, IgA and REG3γ were all increased in NB and BB prefed mice versus BD (P≤.05). Collectively, bean supplementation prior to colitis induction (i.e., mimicking disease relapse) primes the colonic microenvironment to attenuate the severity of the colitis inflammatory phenotype and maintain aspects of epithelial barrier function.

Chickpea supplementation prior to colitis onset reduces inflammation in dextran sodium sulfate-treated C57Bl/6 male mice.

The potential for a chickpea-supplemented diet (rich in fermentable nondigestible carbohydrates and phenolic compounds) to modify the colonic microenvironment and attenuate the severity of acute colonic inflammation was investigated. C57Bl/6 male mice were fed a control basal diet or basal diet supplemented with 20% cooked chickpea flour for 3 weeks prior to acute colitis onset induced by 7-day exposure to dextran sodium sulfate (DSS; 2% w/v in drinking water) and colon and serum levels of inflammatory mediators were assessed. Despite an equal degree of DSS-induced epithelial barrier histological damage and clinical symptoms between dietary groups, biomarkers of the ensuing inflammatory response were attenuated by chickpea pre-feeding, including reduced colon tissue activation of nuclear factor kappa B and inflammatory cytokine production (tumor necrosis factor alpha and interleukin (IL)-18). Additionally, colon protein expression of anti-inflammatory (IL-10) and epithelial repair (IL-22 and IL-27) cytokines were increased by chickpea pre-feeding. Furthermore, during acute colitis, chickpea pre-feeding increased markers of enhanced colonic function, including Relmß and IgA gene expression. Collectively, chickpea pre-feeding modulated the baseline function of the colonic microenvironment, whereby upon induction of acute colitis, the severity of the inflammatory response was attenuated.

Integrated Immunomodulatory Mechanisms through which Long-Chain n-3 Polyunsaturated Fatty Acids Attenuate Obese Adipose Tissue Dysfunction.

Obesity is a global health concern with rising prevalence that increases the risk of developing other chronic diseases. A causal link connecting overnutrition, the development of obesity and obesity-associated co-morbidities is visceral adipose tissue (AT) dysfunction, characterized by changes in the cellularity of various immune cell populations, altered production of inflammatory adipokines that sustain a chronic state of low-grade inflammation and, ultimately, dysregulated AT metabolic function. Therefore, dietary intervention strategies aimed to halt the progression of obese AT dysfunction through any of the aforementioned processes represent an important active area of research. In this connection, fish oil-derived dietary long-chain n-3 polyunsaturated fatty acids (PUFA) in the form of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been demonstrated to attenuate obese AT dysfunction through multiple mechanisms, ultimately affecting AT immune cellularity and function, adipokine production, and metabolic signaling pathways, all of which will be discussed herein.

Fish-oil-derived n-3 polyunsaturated fatty acids reduce NLRP3 inflammasome activity and obesity-related inflammatory cross-talk between adipocytes and CD11b(+) macrophages.

Adipocyte-macrophage cross-talk propagates immune responses in obese adipose tissue (AT). Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) mitigate inflammation, partly through up-regulation of adiponectin; however, specific mechanisms are unclear. We determined if adipocyte-macrophage cross-talk could be mitigated by dietary LC n-3 PUFA and if this was dependent on adiponectin-mediated signaling. We utilized an in vitro co-culture model mimicking the ratio of adipocytes:macrophages in obese AT, whereby 3T3-L1 adipocytes were co-cultured with splenic CD11b(+) macrophages from C57BL/6 mice fed high-fat control (HF-CON; 34% w/w fat) or fish oil diets (HF-FO; 34% w/w fat containing 7.6% w/w FO), as well as mice fed low-fat control (LF-CON; 10% w/w fat) or FO diets (LF-FO; 10% w/w fat containing 3% w/w FO). Co-culture conditions tested effects of soluble mediator-driven mechanisms (trans-well system), cell contact and low-dose lipopolysaccharide (LPS) mimicking acute or chronic inflammatory conditions. HF-FO macrophages from acute LPS-stimulated trans-well co-cultures had decreased mRNA expression of Casp1, Il1ß and Il18, as well as cellular caspase-1 activity compared to HF-CON macrophages (P≤.05). Moreover, adipocytes from acute LPS-stimulated HF-FO co-cultures had decreased caspase-1 activity and decreased IL-1ß/IL-18 levels following chronic LPS pretreatment compared to HF-CON co-cultures (P≤.05). Additionally, in contact co-cultures with adiponectin-neutralizing antibody, the FO-mediated modulation of NFκB activity and decrease in phosphorylated p65 NFκB, expression of NLRP3 inflammasome genes, M1 macrophage marker genes and inflammatory cytokine/chemokine secretion were controlled partly through adiponectin, while cellular caspase-1 activity and IL-1ß/1L-18 levels were decreased independently of adiponectin (P≤.05). LC n-3 PUFA may decrease the intensity of adipocyte-macrophage cross-talk to mitigate obesity-associated pathologies.