Lyticase Facilitates Mycobiome Resolution Without Disrupting Microbiome Fidelity in Primates.

BACKGROUND: Microbiome research has expanded to consider contributions of microbial kingdoms beyond bacteria, including fungi (i.e., the mycobiome). However, optimal specimen handling protocols are varied, including uncertainty of how enzymes utilized to facilitate fungal DNA recovery may interfere with bacterial microbiome sequencing from the same samples. METHODS: With Institutional Animal Care and Use Committee approval, fecal samples were obtained from 20 rhesus macaques (10 males, 10 females; Macaca mulatta). DNA was extracted using commercially available kits, with or without lyticase enzyme treatment. 16S ribosomal RNA (bacterial) and Internal Transcribed Spacer (ITS; fungal) sequencing was performed on the Illumina MiSeq platform. Bioinformatics analysis was performed using Qiime and Calypso. RESULTS: Inclusion of lyticase in the sample preparation pipeline significantly increased usable fungal ITS reads, community alpha diversity, and enhanced detection of numerous fungal genera that were otherwise poorly or not detected in primate fecal samples. Bacterial 16S ribosomal RNA amplicons obtained from library preparation were statistically unchanged by the presence of lyticase. CONCLUSIONS: We demonstrate inclusion of the enzyme lyticase for fungal cell wall digestion markedly enhances mycobiota detection while maintaining fidelity of microbiome identification and community features in non-human primates. In restricted sample volumes, as are common in limited human samples, use of single sample DNA isolation will facilitate increased rigor and controlled approaches in future work.

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Chronic administration of Δ9-tetrahydrocannabinol induces intestinal anti-inflammatory microRNA expression during acute simian immunodeficiency virus infection of rhesus macaques.

UNLABELLED: Recreational and medical use of cannabis among human immunodeficiency virus (HIV)-infected individuals has increased in recent years. In simian immunodeficiency virus (SIV)-infected macaques, chronic administration of Δ9-tetrahydrocannabinol (Δ9-THC) inhibited viral replication and intestinal inflammation and slowed disease progression. Persistent gastrointestinal disease/inflammation has been proposed to facilitate microbial translocation and systemic immune activation and promote disease progression. Cannabinoids including Δ9-THC attenuated intestinal inflammation in mouse colitis models and SIV-infected rhesus macaques. To determine if the anti-inflammatory effects of Δ9-THC involved differential microRNA (miRNA) modulation, we profiled miRNA expression at 14, 30, and 60 days postinfection (days p.i.) in the intestine of uninfected macaques receiving Δ9-THC (n=3) and SIV-infected macaques administered either vehicle (VEH/SIV; n=4) or THC (THC/SIV; n=4). Chronic Δ9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60 days p.i. At 60 days p.i., ∼28% of miRNAs showed decreased expression in the VEH/SIV group compared to none showing decrease in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149, and miR-187, previously been shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator, was confirmed as a direct miR-99b target. A significant increase in NOX4+ crypt epithelial cells was detected in VEH/SIV macaques compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. These results support a role for differential miRNA induction in THC-mediated suppression of intestinal inflammation. Whether similar miRNA modulation occurs in other tissues requires further investigation. IMPORTANCE: Gastrointestinal (GI) tract disease/inflammation is a hallmark of HIV/SIV infection. Previously, we showed that chronic treatment of SIV-infected macaques with Δ9-tetrahydrocannabinol (Δ9-THC) increased survival and decreased viral replication and infection-induced gastrointestinal inflammation. Here, we show that chronic THC administration to SIV-infected macaques induced an anti-inflammatory microRNA expression profile in the intestine at 60 days p.i. These included several miRNAs bioinformatically predicted to directly target CXCL12, a chemokine known to regulate lymphocyte and macrophage trafficking into the intestine. Specifically, miR-99b was significantly upregulated in THC-treated SIV-infected macaques and confirmed to directly target NADPH oxidase 4 (NOX4), a reactive oxygen species generator known to damage intestinal epithelial cells. Elevated miR-99b expression was associated with a significantly decreased number of NOX4+ epithelial cells in the intestines of THC-treated SIV-infected macaques. Overall, our results show that selective upregulation of anti-inflammatory miRNA expression contributes to THC-mediated suppression of gastrointestinal inflammation and maintenance of intestinal homeostasis.

miR-190b is markedly upregulated in the intestine in response to simian immunodeficiency virus replication and partly regulates myotubularin-related protein-6 expression.

HIV replication and the cellular micro-RNA (miRNA) machinery interconnect at several posttranscriptional levels. To understand their regulatory role in the intestine, a major site of HIV/SIV replication, dissemination, and CD4(+) T cell depletion, we profiled miRNA expression in colon following SIV infection (10 acute SIV, 5 uninfected). Nine (four up and five down) miRNAs showed statistically significant differential expression. Most notably, miR-190b expression showed high statistical significance (adjusted p = 0.0032), the greatest fold change, and was markedly elevated in colon and jejunum throughout SIV infection. In addition, miR-190b upregulation was detected before peak viral replication and the nadir of CD4(+) T cell depletion predominantly in lamina propria leukocytes. Interestingly non-SIV-infected macaques with diarrhea and colitis failed to upregulate miR-190b, suggesting that its upregulation was neither inflammation nor immune-activation driven. SIV infection of in vitro-cultured CD4(+) T cells and primary intestinal macrophages conclusively identified miR-190b upregulation to be driven in response to viral replication. Further miR-190b expression levels in colon and jejunum positively correlated with tissue viral loads. In contrast, mRNA expression of myotubularin-related protein 6 (MTMR6), a negative regulator of CD4(+) T cell activation/proliferation, significantly decreased in SIV-infected macrophages. Luciferase reporter assays confirmed MTMR6 as a direct miR-190b target. To our knowledge, this is the first report, which describes dysregulated miRNA expression in the intestine, that identifies a potentially significant role for miR-190b in HIV/SIV pathogenesis. More importantly, miR-190b-mediated MTMR6 downregulation suggests an important mechanism that could keep infected cells in an activated state, thereby promoting viral replication. In the future, the mechanisms driving miR-190b upregulation including other cellular processes it regulates in SIV-infected cells need determination.

Differential Transcriptomic Profiles Following Stimulation with Lipopolysaccharide in Intestinal Organoids from Dogs with Inflammatory Bowel Disease and Intestinal Mast Cell Tumor.

Lipopolysaccharide (LPS) is associated with chronic intestinal inflammation and promotes intestinal cancer progression in the gut. While the interplay between LPS and intestinal immune cells has been well-characterized, little is known about LPS and the intestinal epithelium interactions. In this study, we explored the differential effects of LPS on proliferation and the transcriptome in 3D enteroids/colonoids obtained from dogs with naturally occurring gastrointestinal (GI) diseases including inflammatory bowel disease (IBD) and intestinal mast cell tumor. The study objective was to analyze the LPS-induced modulation of signaling pathways involving the intestinal epithelia and contributing to colorectal cancer development in the context of an inflammatory (IBD) or a tumor microenvironment. While LPS incubation resulted in a pro-cancer gene expression pattern and stimulated proliferation of IBD enteroids and colonoids, downregulation of several cancer-associated genes such as Gpatch4, SLC7A1, ATP13A2, and TEX45 was also observed in tumor enteroids. Genes participating in porphyrin metabolism (CP), nucleocytoplasmic transport (EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) exhibited a similar pattern of altered expression between IBD enteroids and IBD colonoids following LPS stimulation. In contrast, genes involved in anion transport, transcription and translation, apoptotic processes, and regulation of adaptive immune responses showed the opposite expression patterns between IBD enteroids and colonoids following LPS treatment. In brief, the crosstalk between LPS/TLR4 signal transduction pathway and several metabolic pathways such as primary bile acid biosynthesis and secretion, peroxisome, renin-angiotensin system, glutathione metabolism, and arachidonic acid pathways may be important in driving chronic intestinal inflammation and intestinal carcinogenesis.

White button and shiitake mushrooms reduce the incidence and severity of collagen-induced arthritis in dilute brown non-agouti mice.

Exotic mushrooms have been used in ancient Chinese medicine due to their immunomodulatory properties for the treatment and/or prevention of chronic diseases. However, only limited data exist on the health benefits of white button mushrooms (WBM), the most common in the American diet. In the current study, we investigated the effects of WBM and shiitake mushrooms (SM) on collagen-induced arthritis (CIA) using a 2 x 3 factorial design in 8-wk-old female dilute brown non-agouti mice that were fed a control diet (n = 37) or the same diet supplemented with 5% lyophilized WBM or SM (n = 27) for 6 wk. CIA was induced by immunizing mice with 100 µg bovine collagen followed by 50 µg LPS on d 20 post-collagen injection. CIA was assessed by mononuclear cell infiltration, bone erosion, plasma IL-6, TNFα, and intercellular adhesion molecule-1 (sICAM-1) concentrations. Compared with the control diet, WBM and SM tended to reduce the CIA index from 5.11 ± 0.82 to 3.15 ± 0.95 (P = 0.06) (median, 6-9 to 1-2) 31 d post-collagen injection. Whereas 58% of control mice had a CIA index ≥ 7, only 23% of WBM and 29% of SM mice did (P = 0.1). Although both types of mushrooms reduced plasma TNFα (34%, WBM; 64%, SM), only SM increased plasma IL-6 by 1.3-fold (P < 0.05). The CIA index was positively correlated with sICAM1 (r = 0.55; P < 0.05) but negatively correlated with TNFα (r = 0.34; P < 0.05). Whether mushrooms are beneficial for arthritis management remains to be investigated. To our knowledge, this is the first report demonstrating a possible health benefit of WBM in arthritis treatment.

The Acute Host-Response of Turkeys Colonized With Campylobacter coli.

Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) and C. coli are responsible for ~98% of the cases. In turkeys, the ceca are an important anatomical site where Campylobacter asymptomatically colonizes. We previously demonstrated that commercial turkey poults colonized by C. jejuni showed acute changes in cytokine gene expression profiles, and histological intestinal lesions at 2 days post-inoculation (dpi). Cecal tonsils (CT) are an important part of the gastrointestinal-associated lymphoid tissue that surveil material passing in and out of the ceca, and generate immune responses against intestinal pathogens. The CT immune response toward Campylobacter remains unknown. In this study, we generated a kanamycin-resistant C. coli construct (CcK) to facilitate its enumeration from cecal contents after experimental challenge. In vitro analysis of CcK demonstrated no changes in motility when compared to the parent isolate. Poults were inoculated by oral gavage with CcK (5 × 107 colony forming units) or sterile-media (mock-colonized), and euthanized at 1 and 3 dpi. At both time points, CcK was recovered from cecal contents, but not from the mock-colonized group. As a marker of acute inflammation, serum alpha-1 acid glycoprotein was significantly elevated at 3 dpi in CcK inoculated poults compared to mock-infected samples. Significant histological lesions were detected in cecal and CT tissues of CcK colonized poults at 1 and 3 dpi, respectively. RNAseq analysis identified 250 differentially expressed genes (DEG) in CT from CcK colonized poults at 3 dpi, of which 194 were upregulated and 56 were downregulated. From the DEG, 9 significantly enriched biological pathways were identified, including platelet aggregation, response to oxidative stress and negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway. These data suggest that C. coli induced an acute inflammatory response in the intestinal tract of poults, and that platelet aggregation and oxidative stress in the CT may affect the turkey's ability to resist Campylobacter colonization. These findings will help to develop and test Campylobacter mitigation strategies to promote food safety in commercial turkeys.

Microbiota-Related Changes in Unconjugated Fecal Bile Acids Are Associated With Naturally Occurring, Insulin-Dependent Diabetes Mellitus in Dogs.

Diabetes mellitus (DM) in humans has recently been associated with altered intestinal microbiota. The consequences of intestinal dysbiosis, such as increased intestinal permeability and altered microbial metabolites, are suspected to contribute to the host inflammatory state and peripheral insulin resistance. Human diabetics have been shown to have changes in bile acid (BA) metabolism which may be detrimental to glycemic control. The purpose of this study was to examine BA metabolism in dogs with naturally-occurring, insulin-dependent DM and to relate these findings to changes in the intestinal microbiota. A prospective observational study of adult dogs with a clinical diagnosis of DM (n = 10) and healthy controls (HC, n = 10) was performed. The fecal microbiota were analyzed by 16S rRNA gene next-generation (Illumina) sequencing. Concentrations of fecal unconjugated BA (fUBA) were measured using gas chromatography and mass spectrometry. Analysis of bacterial communities showed no significant difference for any of the alpha-diversity measures between DM vs. HC dogs. Principal coordinate analysis based on unweighted Unifrac distance metric failed to show significant clustering between dog groups (ANOSIMUnweighted: R = 0.084; p = 0.114). However, linear discriminate analysis effects size (LEfSe) detected differentially abundant bacterial taxa (α = 0.01, LDA score >2.0) on various phylogenetic levels. While Enterobacteriaceae was overrepresented in dogs with DM, the proportions of Erysipelotrichia, Mogibacteriaceae, and Anaeroplasmataceae were increased in HC dogs. Dogs with DM had increased concentration of total primary fUBA compared to HC dogs (p = 0.028). The concentrations of cholic acid and the cholic acid percentage of the total fUBA were increased (p = 0.028 and p = 0.035, respectively) in the feces of DM dogs relative to HC dogs. The levels of lithocholic acid (both absolute value and percentage of the total fUBA) were decreased (p = 0.043 and p < 0.01, respectively) in DM dogs vs. HC dogs. Results indicate that dogs with DM have both intestinal dysbiosis and associated fUBA alterations. The pattern of dysbiosis and altered BA composition is similar to that seen in humans with Type 2 DM. The dog represents a novel large animal model for advancing translational medicine research efforts (e.g., investigating pathogenesis and therapeutics) in DM affecting humans.

Modulation of gut-specific mechanisms by chronic δ(9)-tetrahydrocannabinol administration in male rhesus macaques infected with simian immunodeficiency virus: a systems biology analysis.

Our studies have demonstrated that chronic Δ(9)-tetrahydrocannabinol (THC) administration results in a generalized attenuation of viral load and tissue inflammation in simian immunodeficiency virus (SIV)-infected male rhesus macaques. Gut-associated lymphoid tissue is an important site for HIV replication and inflammation that can impact disease progression. We used a systems approach to examine the duodenal immune environment in 4- to 6-year-old male rhesus monkeys inoculated intravenously with SIVMAC251 after 17 months of chronic THC administration (0.18-0.32 mg/kg, intramuscularly, twice daily). Duodenal tissue samples excised from chronic THC- (N=4) and vehicle (VEH)-treated (N=4) subjects at ∼5 months postinoculation showed lower viral load, increased duodenal integrin beta 7(+)(ß7) CD4(+) and CD8(+) central memory T cells, and a significant preferential increase in Th2 cytokine expression. Gene array analysis identified six genes that were differentially expressed in intestinal samples of the THC/SIV animals when compared to those differentially expressed between VEH/SIV and uninfected controls. These genes were identified as having significant participation in (1) apoptosis, (2) cell survival, proliferation, and morphogenesis, and (3) energy and substrate metabolic processes. Additional analysis comparing the duodenal gene expression in THC/SIV vs. VEH/SIV animals identified 93 differentially expressed genes that participate in processes involved in muscle contraction, protein folding, cytoskeleton remodeling, cell adhesion, and cell signaling. Immunohistochemical staining showed attenuated apoptosis in epithelial crypt cells of THC/SIV subjects. Our results indicate that chronic THC administration modulated duodenal T cell populations, favored a pro-Th2 cytokine balance, and decreased intestinal apoptosis. These findings reveal novel mechanisms that may potentially contribute to cannabinoid-mediated disease modulation.

White button, portabella, and shiitake mushroom supplementation up-regulates interleukin-23 secretion in acute dextran sodium sulfate colitis C57BL/6 mice and murine macrophage J.744.1 cell line.

Interleukin-23 (IL-23), a cytokine produced primarily by dendritic cells, is involved in host defense against gut pathogens and promotes innate immunity and inflammatory responses through the IL-23/interleukin-17 axis. We previously reported that extracts from edible mushrooms enhanced antimicrobial α-defensin production n HL60 cells. Because IL-23 is involved in defensin production, we hypothesized that edible mushrooms may modulate its secretion and gut inflammation. Eight-week-old C57BL/6 mice were fed the AIN76 diet or the same diet supplemented with 5% white button (WBM), portabella, or shiitake mushrooms. To assess in vivo and in vitro cytokine secretion, 7 to 8 mice per group received 3% dextran sodium sulfate (DSS) in drinking water during the last 5 days of the 6-week feeding period. To delineate the mechanisms by which mushrooms alter IL-23 secretion, J.744.1 cells were incubated with (100 µg/mL) WBM, portabella, and shiitake extracts without and with 100 µg/mL curdlan (a dectin-1 agonist) or 1 mg/mL laminarin (a dectin-1 antagonist). The dectin-1 receptor is a pattern-recognition receptor found in phagocytes, and its activation promotes antimicrobial innate immunity and inflammatory responses. In DSS-untreated mice, mushrooms significantly increased IL-23 plasma levels but decreased those of interleukin-6 (IL-6) (P < .05). In DSS-treated mice, mushroom-supplemented diets increased IL-6 and IL-23 levels (P < .05). Mushroom extracts potentiated curdlan-induced IL-23 secretion, and mushroom-induced IL-23 secretion was not blocked by laminarin in vitro, suggesting the involvement of both dectin-1-dependent and dectin-1-independent pathways. Although all mushrooms tended to increase IL-6 in the colon, only WBM and shiitake tended to increase IL-23 levels. These data suggest that edible mushrooms may enhance gut immunity through IL-23.

Iron deficiency reduces serum and in vitro secretion of interleukin-4 in mice independent of altered spleen cell proliferation.

Iron deficiency, a worldwide public health problem in children and adult women, impairs innate and cell-mediated immunity including interferon-γ secretion. Its effects on interleukin (IL)-4 have not been well investigated. Interleukin-4, a cytokine primarily secreted by TH2 lymphocytes, regulates B-cell proliferation and the switching of immunoglobulin (Ig)M to IgE subtypes; the latter is involved in the defense against helminth infection. Considering the fact that interferon-γ is a potent inhibitor of IL-4, we hypothesize that iron deficiency would increase the secretion of IL-4 and IgE. We measured IL-4 in serum and supernatant of concanavalin A and anti-CD3 antibody-treated spleen cells from iron-deficient, control, pair-fed DBA and C57BL/6 mice (20-24/group) and iron-replete mice for 3, 7, and 14 days (8-13/group). Feeding the low-iron diet (5 ppm vs 50 ppm for the control diet) for 2 months significantly reduced the mean levels of hemoglobin, hematocrit, liver iron stores, thymus weight, and induced splenomegaly in both strains of mice (P < .001). Iron deficiency, and not pair-feeding, reduced plasma IL-4 levels (P < .05), although it did not significantly affect IgE levels. Iron deficiency, especially when associated with thymus atrophy, reduced in vitro IL-4 secretion by activated spleen cells, cell proliferation, and percentage of CD4⁺IL-4⁺ cells (P < .05). Impaired cell proliferation did not fully explain reduced in vitro IL-4 secretion because iron-deficient mice with a normal thymus weight had a normal (3)H-thymidine uptake but decreased supernatant IL-4. It was likely due to low percentage of CD4⁺IL-4⁺. Iron repletion improved IL-4 measurements. Data suggest that iron deficiency has generalized negative effects on T-cell function. Unaltered plasma IgE may be due to other cytokines (ie, IL-13) that also modulate its secretion.

Differential effects of shiitake- and white button mushroom-supplemented diets on hepatic steatosis in C57BL/6 mice.

Shiitake mushrooms (SMs) have been used in Asia for treatment and/or prevention of chronic diseases and hypercholesterolemia. Previously, we observed a diet supplemented with 5% SM resulted in a twofold increase in plasma IL-6 levels in DBA arthritic mice. An elevation in plasma IL-6 has also been implicated in the pathogenesis fatty liver disease. Thus, the aim of this study was to investigate the effect of SM supplemented-diet on hepatic steatosis. In study 1, eight-week old female C57BL/6 mice were randomly assigned to the following groups for 6 weeks: the AIN-93 diet; 5% SM, and 5% white button mushroom (WBM) supplemented diets (12/group). In study 2, mice were fed either the AIN-93 diet or SM (20/group). After 6 weeks, 13 mice fed SM diet were given the AIN93 diet for 8 or 15 days. Unlike other groups, all mice fed the SM diet developed fatty liver (mean histopathology score 4.5 vs <1 in the other groups; p<0.001) without fibrosis and inflammation. Fifteen days post withdrawal of SM completely normalized liver histology. To the best of our knowledge, this is the first report that chronic consumption of SM is associated with the development of fatty liver. The mechanism by which SM causes hepatic steatosis warrants further investigation.