Mononuclear phagocyte morphologic 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 post-translational 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 morphologic response to chemoattractant C5a.

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.

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.

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.

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.

Transient Receptor Potential Vanilloid 4-Dependent Microglial Function in Myelin Injury and Repair.

Microglia are found pathologically at all stages of multiple sclerosis (MS) lesion development and are hypothesized to contribute to both inflammatory injury and neuroprotection in the MS brain. Transient receptor potential vanilloid 4 (TRPV4) channels are widely expressed, play an important role as environmental sensors, and are involved in calcium homeostasis for a variety of cells. TRPV4 modulates myeloid cell phagocytosis in the periphery and microglial motility in the central nervous system. We hypothesized that TRPV4 deletion would alter microglia phagocytosis in vitro and lessen disease activity and demyelination in experimental autoimmune encephalitis (EAE) and cuprizone-induced demyelination. We found that genetic deletion of TRPV4 led to increased microglial phagocytosis in vitro but did not alter the degree of demyelination or remyelination in the cuprizone mouse model of MS. We also found no difference in disease in EAE following global or microglia-specific deletion of Trpv4. Additionally, lesioned and normal appearing white matter from MS brains exhibited similar TRPV4 expression compared to healthy brain tissue. Taken together, these findings indicate that TRPV4 modulates microglial activity but does not impact disease activity in mouse models of MS, suggesting a muted and/or redundant role in MS pathogenesis.

A hydrolyzed lipid blend diet promotes myelination in neonatal piglets in a region and concentration-dependent manner.

The impact of early life nutrition on myelin development is of interest given that cognitive and behavioral function depends on proper myelination. Evidence shows that myelination can be altered by dietary lipid, but most of these studies have been performed in the context of disease or impairment. Here, we assessed the effects of lipid blends containing various levels of a hydrolyzed fat (HF) system on myelination in healthy piglets. Piglets were sow-reared, fed a control diet, or a diet containing 12%, 25%, or 53% HF consisting of cholesterol, fatty acids, monoglycerides, and phospholipid from lecithin. At postnatal day 28/29, magnetic resonance imaging (MRI) was performed to assess changes to brain development, followed by brain collection for microscopic analyses of myelin in targeted regions using CLARITY tissue clearing, immunohistochemistry, and electron microscopy techniques. Sow-reared piglets exhibited the highest overall brain white matter volume by MRI. However, a 25% HF diet resulted in the greatest total myelin density in the prefrontal cortex based on 3D modeling analysis of myelinated filaments. Nodal gap length and g-ratio were inversely correlated with percentage of HF in the corpus callosum, as well as in the PFC and internal capsule for g-ratio, indicating that a 53% HF diet resulted in the thickest myelin per axon and a 0% HF control diet the thinnest in specific brain regions. These findings indicate that HF promoted myelination in the neonatal piglet in a region- and concentration-dependent manner.

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia­induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled­coil­helix­coiled­coil­helix domain­containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). The amplification of the CHCHD2 gene was found to be associated with a decreased patient overall and progression­free survival. The CHCHD2 mRNA levels were increased in high­vs. low­grade glioma, IDH­wt GBMs, and in tumor vs. non­tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII­expressing patient­derived samples. The CRISPR­Cas9­mediated knockout of CHCHD2 in EGFRvIII­expressing U87 cells resulted in an altered mitochondrial respiration and glutathione status, in decreased cell growth and invasion under both normoxic and hypoxic conditions, and in an enhanced sensitivity to cytotoxic agents. CHCHD2 was distributed in both the mitochondria and nuclei of U87 and U87vIII cells, and the U87vIII cells exhibited a greater nuclear expression of CHCHD2 compared to isogenic U87 cells. Incubation under hypoxic conditions, serum starvation and the reductive unfolding of CHCHD2 induced the nuclear accumulation of CHCHD2 in both cell lines. Collectively, the findings of the present study indicate that CHCHD2 mediates a variety of GBM characteristics, and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology.

Influenza A virus infection disrupts oligodendrocyte homeostasis and alters the myelin lipidome in the adult mouse.

BACKGROUND: Recent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior. METHODS: Mice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression. RESULTS: Transcriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability. CONCLUSIONS: These findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process.

Automated identification of piglet brain tissue from MRI images using Region-based Convolutional Neural Networks.

Magnetic resonance imaging is an important tool for characterizing volumetric changes of the piglet brain during development. Typically, an early step of an imaging analysis pipeline is brain extraction, or skull stripping. Brain extractions are usually performed manually; however, this approach is time-intensive and can lead to variation between brain extractions when multiple raters are used. Automated brain extractions are important for reducing the time required for analyses and improving the uniformity of the extractions. Here we demonstrate the use of Mask R-CNN, a Region-based Convolutional Neural Network (R-CNN), for automated brain extractions of piglet brains. We validate our approach using Nested Cross-Validation on six sets of training/validation data drawn from 32 pigs. Visual inspection of the extractions shows acceptable accuracy, Dice coefficients are in the range of 0.95-0.97, and Hausdorff Distance values in the range of 4.1-8.3 voxels. These results demonstrate that R-CNNs provide a viable tool for skull stripping of piglet brains.

Effects of a Lactobacillus fermentation product on the fecal characteristics, fecal microbial populations, immune function, and stress markers of adult dogs.

The objective of this study was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, blood biomarkers, immune function, and serum oxidative stress markers of adult dogs. Thirty adult beagle dogs [23 M, 7 F; mean age = 8.47 ± 2.65 yr old; mean BW = 15.43 ± 4.17 kg] were used in a completely randomized design study. All dogs were fed a basal diet to maintain BW for 5 wk, followed by baseline blood and fecal sample collections. Dogs remained on the same diet, but then were randomly assigned to a placebo (dextrose) or LBFP supplement (Limosilactobacillus fermentum and Lactobacillus delbrueckii). Both treatments were dosed at 4 mg/kg BW via gelatin capsule for 5 wk (n = 15/treatment). Fecal and blood samples were collected at that time. Change from baseline data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Most circulating metabolites and immunoglobulins (Ig) were unaltered by treatment, but LBFP-supplemented dogs had lower changes in serum corticosteroid isoenzyme of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.10), and IgM (P < 0.10) than controls. The change in fecal scores tended to be lower (P = 0.068) in LBFP-supplemented dogs than controls, signifying firmer feces in LBFP-supplemented dogs. Regarding the fecal microbiota, alpha diversity indicators tended to be higher (P = 0.087) in LBFP-supplemented dogs than controls. One fecal bacterial phylum (Actinobacteriota) was altered by treatments, with its relative abundance tending to have a greater (P < 0.10) increase in controls than LBFP-supplemented dogs. Fifteen bacterial genera were altered (P < 0.05 or P < 0.10) by treatments, including relative abundances of fecal Peptoclostridium, Sarcina, and Faecalitalea that had a greater (P < 0.05) increase in controls than LBFP-supplemented dogs. In contrast, relative abundances of fecal Faecalibaculum, Bifidobacterium, and uncultured Butyricicoccaceae had a greater (P ≤ 0.05) increase in LBFP-supplemented dogs than controls. After week 5, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The change in serum superoxide dismutase after transport had a greater (P < 0.0001) increase in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when subjected to stress.

Our objective was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, immune function, and oxidative stress markers of dogs. Thirty adult dogs were used in a completely randomized design study. All dogs were fed a basal diet to maintain body weight for 5 wk and then randomly assigned to a placebo or LBFP supplement for five more weeks. Fecal and blood samples were collected after baseline and treatment phases. Change from baseline data were analyzed statistically. Most blood markers were unaltered by treatment, but LBFP-supplemented dogs had lower changes in liver enzymes and IgM than controls. Change in fecal scores tended to be lower in LBFP-supplemented dogs than controls, signifying firmer feces. Fecal bacterial alpha diversity tended to be higher in LBFP-supplemented dogs than controls. One fecal bacterial phylum and 15 bacterial genera were altered by treatments. After 5 wk, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The increase in serum superoxide dismutase after transport was greater in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when undergoing stress.

α-Tocopherol Transfer Protein-Null Mice with Very Low α-Tocopherol Status Do Not Have an Enhanced Lipopolysaccharide-Induced Acute Inflammatory Response.

Background: The α-tocopherol transfer protein-null (Ttpa-/-) mouse model is a valuable tool for studying the molecular and functional consequences of vitamin E (α-tocopherol, αT) deficiency. Because αT has been associated with reduced oxidative stress and improved immune function, we hypothesized that depleted αT concentration would exacerbate LPS-induced acute inflammatory response in the brain and heart of Ttpa-/- mice fed a vitamin E deficient (VED) diet. Objectives: The objective was to investigate how extremely low αT status, followed by exposure to LPS, altered the acute inflammatory response to LPS in Ttpa-/- and wild-type (Ttpa+/+) mice. Methods: Three-week-old male Ttpa+/+ and Ttpa-/- littermates (n = 36/genotype) ingested a VED diet ad libitum for 4 wk. At week 7, mice received an intraperitoneal LPS (1 or 10 µg/mouse) or saline (control) injection and were killed 4 h postinjection. Brain and heart IL-6 protein concentrations and tissue and serum αT concentrations were measured via ELISA and HPLC with photodiode array detection, respectively. Hippocampal Il-6, Tnf, and Gpx1 gene expression were measured via reverse transcriptase-quantitative polymerase chain reaction, and blood immune cell profiles were measured via a hematology analyzer. Results: αT accumulation in analyzed tissues and serum of Ttpa-/- mice was substantially lower than Ttpa+/+ mice. Circulating white blood cell concentration, particularly lymphocytes, were lower in all LPS groups compared with controls (P < 0.01). The 10 µg LPS groups had elevated IL-6 in the cerebellum and heart compared with controls, confirming an acute inflammatory response (P < 0.01). Hippocampal and heart Il-6 gene expression in the LPS-treated Ttpa-/- mice was upregulated in a dose-dependent manner (P < 0.05). Conclusions: The 10 µg LPS dose enhanced inflammatory markers in the brain, heart, and serum in each genotype but the lower αT status in Ttpa-/- mice did not further impact the acute immune responses.

Endometriosis leads to central nervous system-wide glial activation in a mouse model of endometriosis.

BACKGROUND: Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied. METHODS: Endometriosis was induced in female mice (45-day-old; n = 6-11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning "Weka trainable segmentation" plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated. RESULTS: We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw. CONCLUSION: We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.

Inhibition of inflammatory microglia by dietary fiber and short-chain fatty acids.

Microglia play a vital role maintaining brain homeostasis but can also cause persistent neuroinflammation. Short-chain fatty acids (SCFAs) produced by the intestinal microbiota have been suggested to regulate microglia inflammation indirectly by signaling through the gut-brain axis or directly by reaching the brain. The present work evaluated the anti-inflammatory effects of SCFAs on lipopolysaccharide (LPS)-stimulated microglia from mice fed inulin, a soluble fiber that is fermented by intestinal microbiota to produce SCFAs in vivo, and SCFAs applied to primary microglia in vitro. Feeding mice inulin increased SCFAs in the cecum and in plasma collected from the hepatic portal vein. Microglia isolated from mice fed inulin and stimulated with LPS in vitro secreted less tumor necrosis factor α (TNF-α) compared to microglia from mice not given inulin. Additionally, when mice were fed inulin and injected i.p with LPS, the ex vivo secretion of TNF-α by isolated microglia was lower than that secreted by microglia from mice not fed inulin and injected with LPS. Similarly, in vitro treatment of primary microglia with acetate and butyrate either alone or in combination downregulated microglia cytokine production with the effects being additive. SCFAs reduced histone deacetylase activity and nuclear factor-κB nuclear translocation after LPS treatment in vitro. Whereas microglia expression of SCFA receptors Ffar2 or Ffar3 was not detected by single-cell RNA sequencing analysis, the SCFA transporters Mct1 and Mct4 were. Nevertheless, inhibiting monocarboxylate transporters on primary microglia did not interfere with the anti-inflammatory effects of SCFAs, suggesting that if SCFAs produced in the gut regulate microglia directly it is likely through an epigenetic mechanism following diffusion.

Role of omega-3 endocannabinoids in the modulation of T-cell activity in a multiple sclerosis experimental autoimmune encephalomyelitis (EAE) model.

Epidemiological studies show that omega-3 fatty acid consumption is associated with improved conditions in neurodegenerative diseases such as multiple sclerosis (MS). However, the mechanism of this association is not well understood. Emerging evidence suggests that parent molecules such as docosahexaenoic acid are converted into downstream metabolites that are capable of directly modulating immune responses. In vitro, we found that docosahexaenoyl ethanolamide (DHEA), another dietary component and its epoxide metabolite, reduced the polarization of naïve T-cells toward proinflammatory Th1 and Th17 phenotypes. Furthermore, we identified that DHEA and related endocannabinoids are changing during the disease progression in mice undergoing relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE). In addition, daily administration of DHEA to mice delayed the onset of disease, the rate of relapse, and the severity of clinical scores at relapse in RR-EAE, an animal model of MS. Collectively, these data indicate that DHEA and their downstream metabolites reduce the disease severity in the RR-EAE model of MS and can be potential dietary adjuvants to existing MS therapeutics.

Dietary enrichment of resistant starches or fibers differentially alter the feline fecal microbiome and metabolite profile.

BACKGROUND: Cats are strict carnivores but possess a complex gastrointestinal (GI) microbial community that actively ferments dietary substrates that are not digested and reach the colon. The GI microbiota responses to dietary inclusion of resistant starches versus fibers have not been tested in cats. Thus, our objective was to evaluate the effects of diets enriched in resistant starch or fibers on the fecal characteristics, microbiome, and metabolite profiles of cats. Twelve healthy adult domestic shorthair cats (age = 9.6 ± 4.0 year; body weight = 3.9 ± 1.0 kg) were used in a replicated 3 × 3 Latin square design to test diets that were enriched with: (1) resistant starch (ERS), (2) a fiber-prebiotic-probiotic blend (FPPB), or (3) a fiber-prebiotic-probiotic blend + immune-modulating ingredients (iFPPB). In each 28-day period, 22 days of diet adaptation was followed by fecal and blood sample collection. Fecal samples were used for shotgun metagenomic sequencing. In addition, fecal and blood metabolite measurements and white blood cell stimulation was performed to assess immune function. RESULTS: A total of 1690 bacterial species were identified, with 259 species differing between fiber-rich and ERS treatments. In comparison with fiber-rich treatments that increased diversity and promoted Firmicutes and Bacteroidetes populations, resistant starch reduced microbial diversity and fecal pH, led to a bloom in Actinobacteria, and modified Kyoto Encyclopedia of Genes and Genomes orthology (KO) terms pertaining to starch and sucrose metabolism, fatty acid biosynthesis and metabolism, epithelial cell signaling, among others. Resistant starch also differentially modified fecal metabolite concentrations with relevance to GI and overall host health (increased butyrate; decreased propionate and protein catabolites - branched-chain fatty acids; phenols and indoles; ammonia) and reduced blood cholesterol, which correlated strongly with microbial taxa and KO terms, and allowed for a high predictive efficiency of diet groups by random forest analysis. CONCLUSION: Even though domestic cats and other carnivores evolved by eating low-carbohydrate diets rich in protein and fat, our results demonstrate that the feline microbiome and metabolite profiles are highly responsive to dietary change and in directions that are predictable.

Effects of a Saccharomyces cerevisiae fermentation product-supplemented diet on circulating immune cells and oxidative stress markers of dogs.

Feeding Saccharomyces cerevisiae fermentation product (SCFP) has previously altered fecal microbiota, fecal metabolites, and immune function of adult dogs. The objective of this study was to investigate measures of skin and coat health, changes in circulating immune cell numbers and activity, antioxidant status, and oxidative stress marker concentrations of healthy adult dogs fed a SCFP-supplemented extruded diet. Sixteen adult English Pointer dogs (8 M, 8 F; mean age = 6.7 ± 2.1 yr; mean BW = 25.9 ± 4.5 kg) were used in a randomized crossover design study. All dogs were fed a control diet for 4 wk, then randomly assigned to either the control or SCFP-supplemented diet (0.13% of active SCFP) and fed to maintain BW for 10 wk. A 6-wk washout preceded the second 10-wk experimental period with dogs receiving opposite treatments. After baseline/washout and treatment phases, skin and coat were scored, and pre and postprandial blood samples were collected. Transepidermal water loss (TEWL), hydration status, and sebum concentrations were measured (back, inguinal, ear) using external probes. Oxidative stress and immune cell function were measured by ELISA, circulating immune cell percentages were analyzed by flow cytometry, and mRNA expression of oxidative stress genes was analyzed by RT-PCR. Change from baseline data was analyzed using the Mixed Models procedure of SAS 9.4. Sebum concentration changes tended to be higher (P < 0.10; inguinal, ear) in SCFP-fed dogs than in controls. TEWL change was lower (P < 0.05) on the back of controls, but lower (P = 0.054) on the ear of SCFP-fed dogs. Delayed-type hypersensitivity response was affected by diet and time post-inoculation. Other skin and coat measures and scores were not affected by diet. Changes in unstimulated lymphocytes and stimulated IFN-γ secreting T cells were lower (P < 0.05) in SCFP-fed dogs, while changes in stimulated T cells were lower (P < 0.05) in control-fed dogs. Upon stimulation, the percentage of cytotoxic T cells delta trended lower (P < 0.10) in SCFP-fed dogs. Change in serum superoxide dismutase concentrations was higher (P < 0.05) and change in catalase mRNA expression was lower (P < 0.05) in SCFP-fed dogs. All other measurements of immune cell populations, oxidative stress markers, and gene expression were unaffected by treatment. In conclusion, our data suggest that SCFP positively impacts indicators of skin and coat health of dogs, modulates immune responses, and enhances some antioxidant defense markers.

Saccharomyces cerevisiae fermentation product (SCFP) is a yeast product containing bioactive fermentation metabolites, residual yeast cells, and yeast cell wall fragments. In this study, SCFP was investigated for its impacts on immune health, oxidative stress, and skin and hair coat health in dogs. Using a randomized crossover study design, 16 adult pointer dogs were used to compare changes in immune cell numbers and activity, antioxidant status and oxidative stress marker concentrations, and skin and coat health markers when fed a SCFP-supplemented diet or control diet. Skin sebum concentrations increased in dogs fed SCFP, but transepidermal water loss changes depended on body location (ear, inguinal, or back). Delayed-type hypersensitivity response was affected by diet and time. Changes in unstimulated lymphocytes and stimulated IFN-γ secreting T cells were lower in SCFP-fed dogs, while changes in stimulated T cells were lower in control dogs. Changes in stimulated cytotoxic T cells tended to be lower in SCFP-fed dogs. Change in serum superoxide dismutase concentrations were higher, while change in catalase mRNA expression was lower in SCFP-fed dogs. In conclusion, our data suggest that SCFP positively impacts indicators of skin and coat health of dogs, modulates immune responses, and enhances some key antioxidant defense markers.

Myeloid caspase-8 restricts RIPK3-dependent proinflammatory IL-1ß production and CD4 T cell activation in autoimmune demyelination.

Caspase-8 functions at the crossroad of programmed cell death and inflammation. Here, using genetic approaches and the experimental autoimmune encephalomyelitis model of inflammatory demyelination, we identified a negative regulatory pathway for caspase-8 in infiltrated macrophages whereby it functions to restrain interleukin (IL)-1ß-driven autoimmune inflammation. Caspase-8 is partially activated in macrophages/microglia in active lesions of multiple sclerosis. Selective ablation of Casp8 in myeloid cells, but not microglia, exacerbated autoimmune demyelination. Heightened IL-1ß production by caspase-8-deficient macrophages underlies exacerbated activation of encephalitogenic T cells and production of GM-CSF and interferon-γ. Mechanistically, IL-1ß overproduction by primed caspase-8-deficient macrophages was mediated by RIPK1/RIPK3 through the engagement of NLRP3 inflammasome and was independent of cell death. When instructed by autoreactive CD4 T cells in the presence of antigen, caspase-8-deficient macrophages, but not their wild-type counterparts, released significant amount of IL-1ß that in turn acted through IL-1R to amplify T cell activation. Moreover, the worsened experimental autoimmune encephalomyelitis progression in myeloid Casp8 mutant mice was completely reversed when Ripk3 was simultaneously deleted. Together, these data reveal a functional link between T cell-driven autoimmunity and inflammatory IL-1ß that is negatively regulated by caspase-8, and suggest that dysregulation of the pathway may contribute to inflammatory autoimmune diseases, such as multiple sclerosis.

Dietary Cholesterol Causes Inflammatory Imbalance and Exacerbates Morbidity in Mice Infected with Influenza A Virus.

Influenza is a common cause of pneumonia-induced hospitalization and death, but how host factors function to influence disease susceptibility or severity has not been fully elucidated. Cellular cholesterol levels may affect the pathogenesis of influenza infection, as cholesterol is crucial for viral entry and replication, as well as immune cell proliferation and function. However, there is still conflicting evidence on the extent to which dietary cholesterol influences cholesterol metabolism. In this study, we examined the effects of a high-cholesterol diet in modulating the immune response to influenza A virus (IAV) infection in mice. Mice were fed a standard or a high-cholesterol diet for 5 wk before inoculation with mouse-adapted human IAV (Puerto Rico/8/1934), and tissues were collected at days 0, 4, 8, and 16 postinfection. Cholesterol-fed mice exhibited dyslipidemia characterized by increased levels of total serum cholesterol prior to infection and decreased triglycerides postinfection. Cholesterol-fed mice also displayed increased morbidity compared with control-fed mice, which was neither a result of immunosuppression nor changes in viral load. Instead, transcriptomic analysis of the lungs revealed that dietary cholesterol caused upregulation of genes involved in viral-response pathways and leukocyte trafficking, which coincided with increased numbers of cytokine-producing CD4+ and CD8+ T cells and infiltrating dendritic cells. Morbidity as determined by percent weight loss was highly correlated with numbers of cytokine-producing CD4+ and CD8+ T cells as well as granulocytes. Taken together, dietary cholesterol promoted IAV morbidity via exaggerated cellular immune responses that were independent of viral load.