Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

α-Tocopherol Depletion Exacerbates Lipopolysaccharide-Induced Reduction of Grip Strength.

BACKGROUND: α-Tocopherol (αT) deficiency causes several neurologic disorders, such as spinocerebellar ataxia, peripheral neuropathy, and myopathy. Furthermore, decreased antibody production, impaired ex vivo T cell function, and elevated cytokine production are observed in humans and mice with αT deficiency. Although modeling αT deficiency in animals is challenging, αT depletion can be more readily achieved in α-tocopherol transfer protein-null (Ttpa-/-) mice than wild-type (WT) mice. Thus, the Ttpa-/- mouse model is a useful tool for studying metabolic consequences of low αT status. Optimizing this mouse model and selecting the reliable indicators/markers of deficiency are still needed. OBJECTIVE: Our objective was to assess whether αT depletion alters lipopolysaccharide (LPS)-induced inflammatory response in the brain and/or grip strength used as a proxy for fatigue. METHODS: WT and Ttpa-/- weanling littermates (n = 37-40/genotype) were fed an αT deficient diet ad libitum for 9 wk. Mice were then injected with LPS (10 µg/mouse) or saline (control) intraperitoneally and killed 4 h later. Concentrations of αT in diet and tissues were measured via high-pressure liquid chromatography. Grip strength was evaluated via a grip strength meter apparatus 2 d before and 3.5 h after LPS injection. Cerebellar and serum interleukin-6 (IL-6) concentrations were measured via enzyme-linked immunosorbent assay. RESULTS: αT concentrations in the liver, heart, and adipose tissue of WT mice were higher than Ttpa-/- mice. Although αT was detected in the brain, muscle, and serum of WT mice, it was undetectable in these tissues of Ttpa-/- mice. Cerebellar and serum concentrations of IL-6 were increased in LPS-treated groups but were not significantly affected by genotype. Grip strength was reduced in LPS-treated groups, an effect that was more pronounced in Ttpa-/- mice. CONCLUSIONS: Systemic LPS administration caused an acute inflammatory response with a concomitant decline in grip strength, especially in Ttpa-/- mice. αT depletion appears to exacerbate reductions in grip strength brought on by systemic inflammation.

The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells.

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2's high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2's role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.

Effects of diets supplemented with bioactive peptides on nutrient digestibility, immune cell responsiveness, and fecal characteristics, microbiota, and metabolites of adult cats.

Bioactive peptides (BP) are recognized for their ability to function as antioxidants and maintain lipid stability. They may have positive health effects, including antihypertensive, anti-inflammatory, antimicrobial, osteoprotective, gut health, and immunomodulatory properties, but are poorly tested in cats. Our primary objective was to determine the apparent total tract digestibility (ATTD) of BP-containing kibble diets and assess how the fecal characteristics, metabolites, and microbiota were affected in adult cats. Our secondary objective was to test whether BP could impact blood oxidative stress markers and cytokine concentrations following transport stress. Twelve adult cats (4.83 ± 0.37 yr; 4.76 ± 0.14 kg) were used in a replicated 4 × 4 Latin square design to test four extruded kibble diets: Control (no BP), Chicken (4% chicken BP), Marine1 (2% marine BP), and Marine2 (4% marine BP). Each experimental period lasted 28 d, with a 20-d adaptation phase, 5 d for fecal collection, 2 d for blood collection, and 1 d for transport stress testing (driven in vehicle in individual carriers for 45 min). Salivary cortisol and blood oxidative stress markers and cytokines were measured after transport. Fecal microbiota data were evaluated using 16S rRNA gene amplicon sequencing and QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P < 0.05 being considered significant and P < 0.10 considered trends. No differences were observed in animal health outcomes, with all cats remaining healthy and serum metabolites remaining within reference ranges. Cats fed the Marine2 diet had higher (P < 0.05) ATTD of dry matter (84.5% vs. 80.9%) and organic matter (88.3% vs. 85.8%) than those fed the control diet. The ATTD of protein and energy tended to be higher (P < 0.10) for cats fed the Marine2 diet. Fecal characteristics, metabolites, and bacterial alpha and beta diversity measures were not affected by treatment. However, the relative abundances of six bacterial genera were different (P < 0.05) and two bacterial genera tended to be different (P < 0.10) across treatments. Treatment did not alter salivary cortisol, blood oxidative stress markers, or blood cytokines after transport stress. Our data suggest that BP inclusion may increase nutrient digestibility and modify fecal microbiota and immune measures. More testing is required, however, to determine whether BP may provide additional benefits to cats.

Dietary bioactive peptides (BP) may have positive health effects, but are poorly tested in cats. Our primary objective was to determine the apparent total tract digestibility of BP-containing kibble diets and assess how fecal characteristics, metabolites, and microbiota were affected in adult cats. Our secondary objective was to test whether BP could impact blood oxidative stress markers and cytokines following transport stress. Adult cats were used in a replicated 4 × 4 Latin square design to test four extruded kibble diets containing different BP concentrations. After diet adaptation, fecal and blood samples were collected and transport stress testing was done in each experimental period. All cats remained healthy and serum metabolites remained within reference ranges. Cats fed one of the BP diets had higher dry matter and organic matter digestibilities and tended to have higher protein and energy digestibilities. Fecal characteristics, metabolites, and microbiota diversity measures were not different, but the relative abundances of eight bacterial genera differed or tended to differ across treatments. Treatments did not alter oxidative stress markers after transport stress. Our data suggest that BP inclusion may increase nutrient digestibility and modify fecal microbiota. Further testing is required to determine whether BP provides additional benefits to cats.

Functional properties of Ganoderma lucidum supplementation in canine nutrition.

Ganoderma lucidum (GL) is a mushroom that has been widely used in Asia for its immunostimulatory and anti-inflammatory capacity, which has been hypothesized to be attributed mainly to the recognition of its cell-surface patterns by cells of the immune system present in the gastrointestinal tract, resulting in a cascade of modulatory events. However, the nutraceutical properties of GL have not been tested in dogs. Forty adult beagles were used in a completely randomized design. The objective of the present study was to evaluate the effects of dietary inclusion of GL on peripheral blood mononuclear cells (PBMC; T cells, B cells, monocytes, and natural killers), vaccine response, nutrient digestibility, fecal fermentative end-products, and skin and coat quality of adult dogs. Dogs were fed a commercial dry extruded complete and balanced diet plus GL top-dressed daily upon feeding time. Four experimental treatments were used: 0% GL supplementation (control), 5 mg/kg BW of GL, 10 mg/kg BW of GL, or 15 mg/kg BW of GL. Following a 7 d adaptation to the control diet, dogs were fed their respective treatment diets for 28 d. They were challenged with vaccination of a modified live virus Canine Distemper, Adenovirus Type 1 (Hepatitis), Adenovirus Type 2, Parainfluenza, and Parvovirus and killed Rabies Virus on day 7 with blood collections on days 0, 14, and 28. The inclusion of GL in all dosages was well-accepted by all dogs, with no detrimental effect on macronutrient apparent total tract digestibility. There was a trend that the percentage of major histocompatibility II (MHC-II) from B cells was greater in dogs fed 15 mg/kg of GL (41.91%) compared to the control group (34.63%). The phagocytosis response tended to have treatment-by-time interaction among treatments; dogs fed 15 mg/kg of GL tended to have greater phagocytosis activity on day 28 than dogs from the control group and dogs fed 5 mg/kg of GL. The vaccine-specific serum immunoglobulin G (IgG) concentrations were higher in the group supplemented with 15 mg/kg of GL compared to treatment control 7 d after the vaccination for rabies. These data suggest that the inclusion of GL had no detrimental effects on any analyzed PBMC. Due to changes in immune parameters among treatments, GL may also exert beneficial immunostimulatory effects in healthy adult dogs when provided at a daily dose of 15 mg/ kg BW.

Ganoderma lucidum (GL) is a fungus from which products have become popular in the human food and health industry over the past decade. Due to this, a growing interest in using GL extracts in animal products has also developed. The current study investigated the nutritional properties of GL supplemented to adult beagles in three different inclusion levels in terms of body weight (BW; 5, 10, and 15 mg/kg BW). The results indicated no impact on the overall health, apparent total tract macronutrient digestibility (ATTD), fecal microbial DNA, and skin and coat health. The highlighted results included increased phagocytic activity and vaccine-specific response in the group of dogs supplemented with 15 mg/kg BW.

Respiratory infection with influenza A virus delays remyelination and alters oligodendrocyte metabolism.

Peripheral viral infection disrupts oligodendrocyte (OL) homeostasis such that endogenous remyelination may be affected. Here, we demonstrate that influenza A virus infection perpetuated a demyelination- and disease-associated OL phenotype following cuprizone-induced demyelination that resulted in delayed OL maturation and remyelination in the prefrontal cortex. Furthermore, we assessed cellular metabolism ex vivo, and found that infection altered brain OL and microglia metabolism in a manner that opposed the metabolic profile induced by remyelination. Specifically, infection increased glycolytic capacity of OLs and microglia, an effect that was recapitulated by lipopolysaccharide (LPS) stimulation of mixed glia cultures. In contrast, mitochondrial dependence was increased in OLs during remyelination, which was similarly observed in OLs of myelinating P14 mice compared to adult and aged mice. Collectively, our data indicate that respiratory viral infection is capable of suppressing remyelination, and suggest that metabolic dysfunction of OLs is implicated in remyelination impairment.

Effects of Yeast-Enriched Functionalized Canola Meal Supplementation on Apparent Total Tract Macronutrient Digestibility and Fecal Characteristics, Fecal Microbiota, and Immune Function of Healthy Adult Dogs.

Brewer's dried yeast has a high nutritional value and has long been utilized by the animal feed industry as a source of protein, B-complex vitamins, and minerals. Brewer's dried yeast is also rich in bioactive compounds and may thereby be used as a functional ingredient, providing benefits beyond that of its nutrient content. Canola meal is a high-fiber ingredient that also has unique properties, especially when it is wetted and dried using a proprietary drying system that creates a "functionalized" canola meal. The objective of this experiment was to evaluate the effects of a yeast-enriched functionalized canola meal (FCM) on apparent total tract digestibility (ATTD) and the fecal quality, metabolite concentrations, and microbiota populations, and immune function of healthy adult dogs. Twelve adult female beagles [body weight (BW) = 7.6 ± 0.7 kg; age = 5.8 ± 1.3] were used in a replicated 4×4 Latin square design with 28-d periods. Each experimental period consisted of a 22-d adaptation phase, 5 days of total and fresh fecal collection, and blood collection on the last day. To start, all dogs were fed a basal diet to maintain BW for 14 d. Following fecal and blood collections at baseline (-1 d) to confirm health status, experimental periods began testing the following dietary treatments using a Latin square design experiment: 1) FCM only (no yeast inclusion), 2) FCM + low yeast dose, 3) FCM + medium yeast dose, and 4) FCM + high yeast dose. All treatments were top-dressed onto the basal diet at a rate estimated to be 1% of daily intake (as-is basis). Statistical analysis was performed using the PROC MIXED procedure of SAS with the main effect of treatment and the random effect of dog. Significance was declared at P ≤ 0.05, and trends reported if 0.05 < P ≤ 0.10. Supplementation with yeast-enriched FCM had no significant effect on the ATTD of macronutrients or energy or the fecal characteristics, metabolite concentrations, and microbiota populations of dogs. Additionally, no significant differences were observed in circulating immune cell counts or response to Toll-like receptor agonists among treatments. Our results suggest that the yeast-enriched FCM could be included in canine diets without negatively affecting stool quality, fecal metabolite concentrations, or ATTD. Further research is necessary to determine the effective dose of yeast-enriched FCM, potential mechanisms of action, and other potential implications it has on canine health.