Immunomodulation by cannabidiol in bovine primary ruminal epithelial cells.

BACKGROUND: Ruminant livestock experience a number of challenges, including high concentrate diets, weaning and transport, which can increase their risk of disorders such as ruminal acidosis, and the associated inflammation of the ruminal epithelium. Cannabidiol (CBD), a phytochemical from hemp (Cannabis sativa), is a promising target as a therapy for gastrointestinal inflammation, and may be extremely valuable as either a treatment or prophylactic. However, the effects of CBD in the the ruminant gastrointestinal tract have not been explored, in part due to the restrictions on feeding hemp to livestock. Therefore, the objective of this study was to investigate the immunomodulatory properties of CBD using a model of inflammation in primary ruminal epithelial cells (REC). In addition, CBD dose was evaluated for possible cytotoxic effects. RESULTS: Negative effects on cell viability were not observed when REC were exposed to 10 µM CBD. However, when the dose was increased to 50 µM for 24 h, there was a significant cytotoxic effect. When 10 µM CBD was added to culture media as treatment for inflammation induced with lipopolysaccharide (LPS), expression of genes encoding for pro-inflammatory cytokine IL1B was less compared to LPS exposure alone, and CBD resulted in a down-regulation of IL6. As a pre-treatment, prior to LPS exposure, REC had decreased expression of IL6 and CXCL10 while CBD was present in the media, but not when it was removed prior to addition of LPS. CONCLUSIONS: Results suggest that CBD may reduce cytokine transcription both during LPS-induced inflammation and when used preventatively, although these effects were dependent on its continued presence in the culture media. Overall, these experiments provide evidence of an immunomodulatory effect by CBD during a pro-inflammatory response in primary REC in culture.

Effects of a proinflammatory response on metabolic function of cultured, primary ruminal epithelial cells.

Inflammation of ruminal epithelium may occur during ruminal acidosis as a result of translocation and interaction of ruminal epithelial cells (REC) with molecules such as lipopolysaccharide (LPS). Such inflammation has been reported to alter cellular processes such as nutrient absorption, metabolic regulation, and energy substrate utilization in other cell types but has not been investigated for REC. The objectives of this study were to investigate the effects of LPS on metabolism of short-chain fatty acids by primary REC, as well as investigating the effects of media containing short-chain fatty acids on the proinflammatory response. Ruminal papillae from 9 yearling Speckle Park beef heifers were used to isolate and culture primary REC. Cells were grown in minimum essential medium (MEM) for 12 d before use and then reseeded in 24-well culture plates. The study was conducted as a 2 × 2 factorial, where cells were grown in unaltered MEM (REG) or medium containing 2 mM butyrate and 5 mM propionate (SCFA) with (50,000 EU/mL; +LPS) or without LPS (-LPS) for 24 h. Supernatant samples were collected for analysis of glucose and SCFA consumption. Cells were collected to determine the expression of mRNA for genes associated with inflammation (TNF, IL1B, CXCL2, CXCL8, PTGS2, and TLR4), purinergic signaling (P2RX7, ADORAB2, and CD73), nutrient transport [SLC16A1 (MCT1), SLC16A3 (MCT4), SLC5A8, and MCU], and cell metabolism [ACAT1, SLC2A1 (GLUT1), IGFBP3, and IGFBP5]. Protein expression of TLR4 and ketogenic enzymes (BDH1 and HMGCS1) were also analyzed using flow cytometry. Statistical analysis was conducted with the MIXED model of SAS version 9.4 (SAS Institute Inc., Cary, NC) with medium, LPS exposure, and medium × LPS interaction as fixed effects and animal within plate as a random effect. Cells tended to consume more glucose when exposed to LPS as opposed to no LPS exposure (31.8 vs. 28.7 ± 2.7), but consumption of propionate and butyrate was not influenced by LPS. Expression of TNF and IL1B was upregulated when exposed to LPS, and expression of CXCL2 and CXCL8 increased following LPS exposure with SCFA (medium × LPS). For cells exposed to LPS, we found a downregulation of ACAT1 and IGFBP5 and an upregulation of SLC2A1, SLC16A3, MCU, and IGFBP3. Medium with SCFA led to greater expression of MCU. SLC16A1 was upregulated in cells incubated with SCFA and without LPS compared with the other groups. Protein expression of ketogenic enzymes was not affected; however, BDH1 mean fluorescence intensity (MFI) expression tended to be less in cells exposed to LPS. These data are interpreted to indicate that when REC are exposed to LPS, they may increase glucose metabolism. Moreover, transport of solutes was affected by SCFA in the medium and by exposure to LPS. Overall, the results suggest that metabolic function of REC in vitro is altered by a proinflammatory response, which may lead to a greater glucose requirement.

Effects of lipopolysaccharide exposure on the inflammatory response, butyrate flux, and metabolic function of the ruminal epithelium using an ex vivo model.

Acidotic conditions in the rumen have been associated with compromised barrier function of the ruminal epithelium and translocation of microbe-associated molecular patterns (MAMP) such as lipopolysaccharide (LPS). Interaction of MAMP with the ruminal epithelium may also induce a local proinflammatory response. The aim of this study was to evaluate the potential proinflammatory response of the ruminal epithelium following LPS exposure in Ussing chambers, to investigate whether LPS exposure affects the flux and metabolism of butyrate. Ruminal epithelial tissue from 9 Holstein bull calves were mounted into Ussing chambers and exposed to 0, 10,000, 50,000, or 200,000 endotoxin units (EU)/mL LPS for a duration of 5 h. Radiolabeled 14C-butyrate (15 mM) was added to the mucosal buffer to assess the mucosal-to-serosal flux of 14C-butyrate. Additional Ussing chambers, without radioisotope, were exposed to either 0 or 200,000 EU/mL LPS and were used to measure the release of ß-hydroxybutyrate (BHB) and IL1B into the buffer, and to collect epithelial tissue for analysis of gene expression. Genes associated with inflammation (TNF, IL1B, CXCL8, PTGS2, TGFB1, TLR2, TLR4), nutrient transport (MCT1, MCT4, SLC5A8, GLUT1), and metabolic function (ACAT1, BDH1, MCU, IGFBP3, IGFBP5) were selected and analyzed using quantitative real-time PCR. Butyrate flux was not significantly affected by LPS exposure; however, we detected a tendency for the mucosal-to-serosal butyrate flux to increase linearly with LPS dose. Bidirectional releases of BHB and IL1B were not affected by LPS exposure. Expression of PTGS2, TGFB1, TLR4, and MCU were downregulated following exposure to LPS ex vivo. We detected no effects on the expression of genes associated with nutrient transport. The results of the present study are interpreted to indicate that, although the inflammatory response of the ruminal epithelium was slightly suppressed, exposure to LPS may have altered metabolic function.

Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells.

The objective of this study was to investigate whether cultured ruminal epithelial cells (REC) responded to lipopolysaccharide (LPS) stimulation and determine whether LPS induced a proinflammatory response. Primary bovine REC were isolated and grown in culture for 2 studies. In study 1, REC were isolated from Holstein bull calves (n = 8) and grown in culture for 10 to 12 d. Cells were then exposed to 0, 10,000, 50,000, or 200,000 endotoxin (E)U/mL of LPS (Escherichia coli O55:B5) for either 6 or 24 h. The effect of LPS exposure on cell viability was analyzed by flow cytometry using a propidium iodide stain. In study 2, cells were isolated from Holstein bull calves (n = 5) and yearling beef heifers (n = 4). Cells were exposed to either 1,000 or 50,000 EU/mL of LPS using the following conditions: (1) medium alone time-matched controls, (2) 12-h LPS exposure, (3) 24 h of LPS exposure, (4) 36 h of LPS exposure, (5) 12 h of LPS exposure followed by LPS removal for 24 h before restimulating with LPS for an additional 12 h (RPT), and (6) 12 h of LPS exposure followed by LPS removal for 36 (RVY). For both experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF and IL1B), chemokines (CXCL2 and CXCL8), a lipid mediator (PTGS2), and growth factor-like cytokines (CSF2 and IL7). In study 1, LPS exposure did not negatively affect cell viability. Treatment of cells with LPS resulted in increased transcript abundance for all genes analyzed. The TLR2, IL7, and TLR4 had a greater magnitude of change at 6 h compared with 24 h. Quadratic expression patterns were detected for TNF, IL1B, CXCL2, CXCL8, and CSF2. These results suggested that REC increase expression of proinflammatory genes following exposure to LPS. In study 2, all genes analyzed were upregulated in a quadratic manner following exposure to LPS for different time intervals. The TLR4, TNF, CXCL2, CXCL8, CSF2, and IL7 gene expression was significantly greater after a single 12 h of LPS exposure than after RPT exposure, suggesting repeated exposure of REC to LPS may induce a tolerogenic effect. When LPS was removed from the medium (RVY), transcript abundance for all genes analyzed decreased and expression of TLR2, TLR4, and IL7 returned to baseline levels, suggesting REC recovered following exposure to LPS. Overall, the data suggest cultured REC respond to LPS stimulation by increasing transcription of proinflammatory genes and this transcriptional response was influenced by the dose, duration, and frequency of LPS exposure.

Effect of heat-treated canola meal and glycerol inclusion on performance and gastrointestinal development of Holstein calves.

The objectives of this study were to assess the effect of using heat-treated canola meal (CM) and glycerol inclusion in starter mixtures on starter intake, growth, and gastrointestinal tract development in Holstein bull calves. In the first study, a protocol for the heat treatment of CM was evaluated by comparing commercial CM that was exposed to 0, 100, 110, or 120°C of heat treatment for 10 min. Following heat treatment, in situ crude protein (CP) ruminal degradability and estimated intestinal CP digestibility were assessed. It was observed that the degradable fractions of dry matter and CP in CM decreased linearly with increasing temperature of heat treatment. The estimated intestinal CP digestibility was greatest when CM was heated to 110°C. In the second study, 28 bull calves were used in a randomized complete block design. Calves were fed pelleted starters containing CM or CM that was heat-treated to 110°C for 10 min. Diets also contained 0 or 5% glycerol on a dry matter basis. The study lasted 51 d, ending on the first day of weaning. Starter intake, average daily gain (ADG), ruminal short-chain fatty acid concentrations, morphology of the rumen and small intestine, gene expression (MCT1, GPR41, GPR43, UTB, AQP3, PEPT1, PEPT2, ATB0+, and EAAC1) in the ruminal, jejunal, and ileal epithelium, and brush border enzyme activities in the duodenum, jejunum, and ileum were investigated. Few interactions between heat-treated CM and glycerol inclusion were observed. Feeding heat-treated CM did not affect starter intake. However, feeding heat-treated CM to calves tended to reduce ADG and decreased the weight of ruminal and jejunal tissue. Heat treatment did not affect gene expression or brush border enzyme activities in the small intestine. Glycerol inclusion tended to increase cumulative starter intake and increased cumulative body weight gain. Use of glycerol reduced ruminal pH and increased the concentration of ruminal short-chain fatty acids. Additionally, glycerol inclusion increased abomasal, duodenal, jejunal, and cecal digesta weights and tended to increase the weight of the jejunal tissue. Glycerol supplementation tended to downregulate the expression of MCT1 in the ruminal epithelium, and upregulated the expression of MCT1 in the epithelium of proximal jejunum. In conclusion, heat treatment of CM may negatively affect calf growth and gastrointestinal tract development. Glycerol inclusion may increase starter intake, ADG, ruminal fermentation, and intestinal development in calves when CM is used as a main source of protein in pelleted starter mixture.

Potential for a localized immune response by the ruminal epithelium in nonpregnant heifers following a short-term subacute ruminal acidosis challenge.

The aim of this study was to investigate whether the ruminal epithelium activates a local inflammatory response following a short-term subacute ruminal acidosis (SARA) challenge. Seven ruminally cannulated, nonpregnant, nonlactating beef heifers, fed a baseline total mixed ration (TMR) with 50:50 forage-to-concentrate ratio, were used in a crossover design with 2 periods and 2 treatments: SARA and control (CON). Induction of SARA included feed restriction (25% of dry matter intake [DMI] for 24 h) followed by a grain overload (30% of baseline DMI) and provision of the full TMR; whereas, the CON group received the TMR ad libitum. Ruminal pH was recorded using indwelling probes, and ruminal lipopolysaccharide (LPS) concentration was measured daily following the challenge until d 6. Biopsies of ruminal papillae from the ventral sac were collected on d 2 and 6 after the grain overload. Transcript abundance of genes associated with acute inflammation was measured by quantitative real-time PCR, normalized to the geometric mean of 3 stable housekeeping genes. Target genes included toll-like receptor-2 (TLR2), TLR4, TLR9, tumor necrosis factor-α (TNFA), prostaglandin endoperoxide synthase-1 (PTGS1), PTGS2 transforming growth factor ß-1 (TGFB1), and 4 intermediate enzymes of leukotriene synthesis (ALOX5, ALOX5AP, LTA4H, and LTC4S). Protein localization and expression of TLR4 were quantified by image analysis of fluorescence intensity. Statistical analysis was performed using as a crossover design with fixed effects of treatment, day, and the treatment × day interaction with the random effect of day within period. Ruminal pH was below 5.6 for 4.5 h/d and below 5.8 for 6.9 h/d in the SARA group compared with 22 and 72 min/d, respectively, for CON. Ruminal LPS concentration peaked on d 2 in SARA heifers at 51,481 endotoxin units (EU)/mL compared with 13,331 EU/mL in CON. Following grain overload, small but statistically significant decreases in the transcriptional abundance of TLR2, TLR4, TNF, PTGS2, ALOX5, and ALOX5AP were seen in SARA versus CON heifers. A functionally relevant decrease in TLR4 expression in SARA heifers compared with CON was confirmed by a decrease in fluorescence intensity of the corresponding protein following immunohistofluorescent staining of papillae. The study results indicate a suppression of the inflammatory response in the ruminal epithelium and suggest that the response is tightly regulated, allowing for tissue recovery and return to homeostasis following SARA.