Conventionalized Research Articles

Absence of gut microbiota reduces neonatal survival and exacerbates liver disease in Cyp2c70-deficient mice with a human-like bile acid composition.

Mice with deletion of Cyp2c70 have a human-like bile acid composition, display age- and sex-dependent signs of hepatobiliary disease and can be used as a model to study interactions between bile acids and the gut microbiota in cholestatic liver disease. In the present study, we rederived Cyp2c70-/- mice as germ-free (GF) and colonized them with a human or a mouse microbiota to investigate whether the presence of a microbiota can be protective in cholangiopathic liver disease associated with Cyp2c70-deficiency. GF Cyp2c70-/- mice showed reduced neonatal survival, liver fibrosis, and distinct cholangiocyte proliferation. Colonization of germ-free breeding pairs with a human or a mouse microbiota normalized neonatal survival of the offspring, and particularly colonization with mouse microbiota from a conventionally raised mouse improved the liver phenotype at 6-10 weeks of age. The improved liver phenotype in conventionalized (CD) Cyp2c70-/- mice was associated with increased levels of tauro-ursodeoxycholic acid (TUDCA) and UDCA, resulting in a more hydrophilic bile acid profile compared with GF and humanized Cyp2c70-/- mice. The hydrophobicity index of biliary bile acids of CD Cyp2c70-/- mice was associated with changes in gut microbiota, liver weight, liver transaminases, and liver fibrosis. Hence, our results indicate that neonatal survival of Cyp2c70-/- mice seems to depend on the establishment of a gut microbiota at birth, and the improved liver phenotype in CD Cyp2c70-/- mice may be mediated by a larger proportion of TUDCA/UDCA in the circulating bile acid pool and/or by the presence of specific bacteria.

The gut microbiome restores intrinsic and extrinsic nerve function in germ‐free mice accompanied by changes in calbindin

The microbiome is essential for normal myenteric intrinsic primary afferent neuron (IPAN) excitability. These neurons control gut motility and modulate gut-brain signaling by exciting extrinsic afferent fibers innervating the enteric nervous system via an IPAN to extrinsic fiber sensory synapse. We investigated effects of germ-free (GF) status and conventionalization on extrinsic sensory fiber discharge in the mesenteric nerve bundle and IPAN electrophysiology, and compared these findings with those from specific pathogen-free (SPF) mice. As we have previously shown that the IPAN calcium-dependent slow afterhyperpolarization (sAHP) is enhanced in GF mice, we also examined the expression of the calcium-binding protein calbindin in these neurons in these different animal groups. IPAN sAHP and mesenteric nerve multiunit discharge were recorded using ex vivo jejunal gut segments from SPF, GF, or conventionalized (CONV) mice. IPANs were excited by adding 5 μM TRAM-34 to the serosal superfusate. We probed for calbindin expression using immunohistochemical techniques. SPF mice had a 21% increase in mesenteric nerve multiunit firing rate and CONV mice a 41% increase when IPANs were excited by TRAM-34. For GF mice, this increase was barely detectable (2%). TRAM-34 changed sAHP area under the curve by -77 for SPF, +3 for GF, or -54% for CONV animals. Calbindin-immunopositive neurons per myenteric ganglion were 36% in SPF, 24% in GF, and 52% in CONV animals. The intact microbiome is essential for normal intrinsic and extrinsic nerve function and gut-brain signaling.

Sa1767 STAT3 Activation in CD4+ Lymphocytes Shapes Epithelial Host Defense During Infectious Colitis

Background: The commensal microbiota are known to regulate the activity of neutrophils and other leukocytes in the intestine, but the impact of the microbiota on systemic neutrophil activity remains unknown. Serum amyloid A (Saa) is a circulating HDL-associated apolipoprotein and acute phase protein. Saa genes are expressed by multiple tissues and cell types and are markedly induced by diverse stimuli including the gut microbiota. However, the precise roles of Saa in inflammation remain elusive because both pro-and anti-inflammatory actions have been reported. Aim: Assess the effects of microbiota colonization and Saa induction on systemic neutrophil development and function. Methods: The effect of the microbiota on neutrophil localization and migration was evaluated using germ-free (GF) and conventionalized (CONVD) transgenic zebrafish expressing GFP in neutrophils (Tg(mpx:GFP)) Flow cytometry was used to quantify and sort GFP(+) neutrophils for qRTPCR ofmpxmRNA levels. Neutrophil migrationwas assessed using tracking analysis of GFP(+) neutrophils using Volocity. Neutrophil recruitment was assessed using a tail-wounding assay. Morpholinos targeting saa were used on GF and CONVD fish to evaluate the role of saa on neutrophil biology. In vitro studies were performed using PAC-2 zebrafish embryonic fibroblast cells and peritoneal isolated murine neutrophils. NF-κB pathway activation was measured in SAA stimulated PAC-2 cells by western blot and through transfection of a pikbaa:Luc reporter. Murine neutrophil migration in response to SAA in the presence of an NF-κB inhibitor was assessed using a transwell system. Results: The presence of a microbiota resulted in increased neutrophil number (~3 fold, p<0.05) and myeloperoxidase expression (~5 fold, p<0.05)), and altered neutrophil localization and migratory behaviors (~2 fold increased velocity and increased directional migration). These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to the injury site. Morpholino knockdown experiments revealed saamediates microbial stimulation of tissue-specific neutrophil migratory behaviors. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB (~3 fold, p<0.001), and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression.Conclusions:These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signaling axis in mediating neutrophil migratory responses.

Sa1765 Defective Autophagy Contributes to Impaired Bacterial Clearance of SAMP1/YITFC Mice

Background: The commensal microbiota are known to regulate the activity of neutrophils and other leukocytes in the intestine, but the impact of the microbiota on systemic neutrophil activity remains unknown. Serum amyloid A (Saa) is a circulating HDL-associated apolipoprotein and acute phase protein. Saa genes are expressed by multiple tissues and cell types and are markedly induced by diverse stimuli including the gut microbiota. However, the precise roles of Saa in inflammation remain elusive because both pro-and anti-inflammatory actions have been reported. Aim: Assess the effects of microbiota colonization and Saa induction on systemic neutrophil development and function. Methods: The effect of the microbiota on neutrophil localization and migration was evaluated using germ-free (GF) and conventionalized (CONVD) transgenic zebrafish expressing GFP in neutrophils (Tg(mpx:GFP)) Flow cytometry was used to quantify and sort GFP(+) neutrophils for qRTPCR ofmpxmRNA levels. Neutrophil migrationwas assessed using tracking analysis of GFP(+) neutrophils using Volocity. Neutrophil recruitment was assessed using a tail-wounding assay. Morpholinos targeting saa were used on GF and CONVD fish to evaluate the role of saa on neutrophil biology. In vitro studies were performed using PAC-2 zebrafish embryonic fibroblast cells and peritoneal isolated murine neutrophils. NF-κB pathway activation was measured in SAA stimulated PAC-2 cells by western blot and through transfection of a pikbaa:Luc reporter. Murine neutrophil migration in response to SAA in the presence of an NF-κB inhibitor was assessed using a transwell system. Results: The presence of a microbiota resulted in increased neutrophil number (~3 fold, p<0.05) and myeloperoxidase expression (~5 fold, p<0.05)), and altered neutrophil localization and migratory behaviors (~2 fold increased velocity and increased directional migration). These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to the injury site. Morpholino knockdown experiments revealed saamediates microbial stimulation of tissue-specific neutrophil migratory behaviors. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB (~3 fold, p<0.001), and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression.Conclusions:These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signaling axis in mediating neutrophil migratory responses.

Screening for Intestinal Microflora Influencing Superoxide Dismutase Activity in Mouse Cecal Mucosa

ABSTRACTWe have suggested that intestinal microflora reduces the activity of the antioxidant enzyme superoxide dismutase (SOD) in the mouse cecal mucosa. In this study, gnotobiotic mice were used to examine the species of intestinal microflora influencing SOD activity in the cecal mucosa. The total SOD activity in the cecal mucosa of each germ-free (GF), gnotobiotic mouse with Escherichia coli, Lactobacillus and Bacteroides was significantly higher than that in the cecal mucosa of gnotobiotic mice with chloroform-treated feces (CHF), conventionalized (CVz) mice and conventional (CV) mice (P<0.05). In addition, CuZnSOD mRNA expression showed similar tendencies. Our results suggest that the antioxidant defense status in the cecal mucosa is influenced by CHF inoculation.

Delayed bacterial colonization of the gut alters the host immune response to oral sensitization against cow's milk proteins

Cow's milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cow's milk β-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age. Oral sensitization to cow's milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to αS1- and κ-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice. Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.

Bacterial signaling through Nod2/GSK3b protects the small intestine against ischemia‐reperfusion induced injury in mice

Ischemia/reperfusion (I/R) injury represents an important mucosal injury and is one of the leading causes of death in critically ill patients.AimTo investigate the impact of bacteria/Nod2 signaling in I/R‐mediated injury.MethodsThe human colonic HCT‐116 (expressing endogenous Nod2) cells and rat IEC‐6 cells infected with Ad5Nod2 viral vector were used to study muramyl dipeptide (MDP; 50 ug/mL) signaling. GSK3β (Ser9), histone 3 (Ser10), β‐catenin phosphorylation levels were determined by Western blot or immunohistochemistry analysis. Germ‐free (GF), conventionalized (CNV), Nod2 knock‐out (Nod2−/−) and wild‐type (WT) mice (C57BL/6; 6–8 mice/group) were subjected to intestinal I/R by partial occlusion of the 2 distal branches of the superior mesenteric artery for 60 min, followed by 4 h reperfusion. Proliferation was determined using Ki67 IHC staining.ResultsGSK3β phosphorylation and nuclear accumulation of β‐catenin is induced within 60 min in MDP‐stimulated HCT‐116 cells and Ad5Nod2‐infected IEC‐6 cells. I/R‐induced ileal injury worsen in GF mice compared to CNV mice (1.5±0.22 vs 0.7±0.20, p&lt;0.05) which associated with shorten crypt/villin length (11.9±0.19 μm vs 18.1±0.21 μm, p&lt;0.01). Importantly, I/R‐induced ileal injury is more pronounced in Nod2−/− mice compared to WT mice (1.9±0.23 vs 0.9±0.29, p&lt;0.05), with concomitant shorten crypt/villi length (11.8±0.17 μm vs 18.0±0.16 μm, p&lt;0.05) and decreased IL‐6 mRNA accumulation (18.5±5.7 vs. 54.7±9.4). This increased injury response was accompanied by reduced Ki67 staining, GSK3β and H3 phosphorylation.ConclusionBacteria/Nod2 signaling to GSK3β/β‐catenin pathway represents an important host response to the microbiota and may protect the small intestine against I/R‐induced injury.

Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs

Porcine proglucagon mRNA sequence was determined by designing primers based on homologous regions in bovine and human genes. The porcine sequence shared 90% nucleotide identity with human proglucagon; however, predicted Ser159Arg and Leu160Lys substitutions were observed. This newly identified sequence suggests that the intestinal trophic peptide, glucagon-like peptide 2 (GLP-2), is actually 35 amino acids in the pig rather than 33 amino acids, as previously reported. To determine the effect of different bacteria on intestinal proglucagon gene expression, 16 piglets were reared in gnotobiotic isolators maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized (CV) in two separate experiments for 13 d. Cultured cecal contents confirmed microbial status with the exception of GF pigs in exp. 2, which were contaminated with Staphylococcus epidermidis (SE). Mean fold differences in ileal proglucagon expression (CV set to 1) were 1.0ab, 1.6a, 0.7b, 1.0ab for GF, LF, EC and CV in exp. 1 and 4.0a, 2.0b, 0.6c, 1.0c for SE, LF, EC and CV groups in exp. 2, respectively. This study, for the first time, accurately describes the full nucleotide sequence for porcine proglucagon, and shows that proglucagon expression is differentially regulated by bacteria colonizing intestine of neonatal piglets. Key words: Proglucagon, gnotobiotic, pig, gene expression, intestine

Inhibition of polyposis in the small intestine of BALB/c mice by intestinal bacteria

The incidence of polyposis and the number of polyps per mouse were significantly lower in conventionalized (CVz) mice than in germ-free (GF) mice. There was no significant difference in the average number of polyps between GF and gnotobiotic (GB) mice monoassociated with the various strains of intestinal bacteria. However, the incidence of polyposis and the number of polyps per mouse were significantly lower for mice associated with either chloroform-resistant bacteria (CRB) or fusiform bacteria (FB) than for GF mice This study demonstrated that polyposis was suppressed by FB and CRB in the small intestine of BALB/c mice.

Changes in body composition of germ-free and conventional chickens during starvation

1. 1. Changes in body composition during starvation were compared between germ-free (GF) and conventionalized (CVL) chicks in experiment 1. At 8 days of age, the GF birds were divided into two groups, i.e. GF and CVL groups. The CVL birds were inoculated with faeces from conventionally reared birds. Until 14 days of age, both birds were fed a diet ad lib, and thereafter starved for 6 days. 2. 2. Nitrogen loss during starvation was significantly lower in CVL birds, though the reverse was true for water loss. Fasting heat production was comparable between two environments. 3. 3. Influence of the gut microflora on body weight and nitrogen losses during starvation was investigated in birds prefed diets high or low in dietary protein in experiment 2. 4. 4. No significant effect of the gut microflora was observed in body weight and nitrogen losses. Body weight was severely reduced in birds prefed the high protein diet and nitrogen loss was lower in birds prefed the low protein diet.

Identification and characterization of rat intestinal lamina propria cells: consequences of microbial colonization

Germ-free (GF) animals exhibit an abnormally diminished, cell-mediated immune response which can be rapidly normalized by bacterial colonization of the intestine. This conventionalization suggests that the development and/or regulation of the immune system is dependent upon intestinal bacteria or their products. Here we consider the ontogeny of gut-associated lymphoid tissue (GALT) immunocytes by isolating and characterizing the intestinal lamina propria cells (LPC) of GF rats responding to bacterial colonization or an irrelevant protein antigen, and compared to LPC of specific pathogen-free (SPF) rats which were conventionalized (CV) from birth. Isolation of cells was accomplished by successive EDTA washings of small intestine to remove the epithelium, and enzymatic digestion of the tissue generating single-cell suspensions. Resulting cell suspensions were characterized by monoclonal antibodies directed against leukocyte epitopes using flow cytometry. Functional characterization was measured by the tritiated thymidine proliferation assay with concanalin A (Con A) and lipopolysaccharide (LPS) as co-stimulators. Germ-free and SPF rats had fewer total LPC than CV rats. Antibody staining revealed that GF rats had fewer total leukocytes than CV and SPF rats, and that CV rats had a greater percentage of T-cells and cells positive for the C3 receptor than GF rats. Co-stimulation of LPC with mitogens only increased proliferation of cells from CV rats compared to GF and SPF rats. In addition, spleen cells from CV rats demonstrated significantly enhanced proliferative responses compared to spleen cells from GF rat and were more analogous to spleen cells from SPF rats in their ability to proliferate in vitro, with and without mitogens. We conclude that T-cells and CD35-positive (C3BR+) cells are recruited and/or proliferate in response to intestinal bacteria and/or their products, and that this results in the induction of immune competency.

Ammonia in the intestinal contents from germfree rats

The values of pH and the concentrations of nitrogen (N) and ammonia in the middle part of the small intestinal and cecal contents of germfree (GF) and conventionalized (CVZ) seven-week-old rats were compared. The pH of the small intestinal and cecal contents of GF rats was higher than that of CVZ rats. There was no difference in total N per fresh weight in contents from the middle part of the small intestine between GF and CVZ, whereas total N per fresh weight of the cecal contents was higher in CVZ than in GF rats. The ammonia concentrations per fresh weight or per total N in the intestinal and cecal contents of CVZ rats were higher than those of GF rats.

Alteration in sensitivity of transmural electrical response to propionate in rat colon after chronic luminal infusion of short-chain fatty acids

The colon is always exposed to abundant short-chain fatty acids (SCFA) produced by gut fermentation. In order to know an effect of chronic load of SCFA on colonic functions, we studied that the acute and chronic effects of SCFA on transmural potential difference (p.d.) across the proximal colon of germ-free (GF), gnotobiotic (GB) and conventionalized (CV) rats in vivo . Intravenous administration of SCFA (acute effect), such as propionate, butyrate, valerate or caproate, caused a transient increase in the p.d. The acute effects of propionate were studied in detail. The dose-response curve of CV rats shifted markedly to the right compared to that of GH rats, suggesting that CV rats were less sensitive to the acute effects of propionate than GF rats. Decreased sensitivity also appeared in GB rats (monocontamination with Fusobacterium varium ). By chronic luminal infusion of isotonic sodium propionate or butyrate (25.5 ml/day) into the proximal colon of GF rats for 7 days (chronic effect), the acute effects of propionate were reduced. Atropine reduced the p.d. increment produced by propionate and shifted the dose-response curve of propionate to the right. These results suggest that chronic luminal load of SCFA resulted in a type of chronic refractoriness.

Acute Choline Deficiency in Germfree, Conventionalized and Open-Animal-Room Rats: Effects of neomycin, chlortetracycline, vitamin B12 and coprophagy prevention

Acute choline deficiency was studied in germfree (GF), conventionalized (CONV) and open-animal-room (OAR) rats. With diet A (low cystine, low cholesterol), no rats died; nephropathy and liver fat were less in the GF rats. Liver cholesterol (measured only in OAR rats) increased. Neomycin was without effect in GF and OAR rats. Vitamin B12 lessened the nephropathy and liver fat in only OAR rats; cholesterol was not measured. GF rats fed diet B (high cystine, high cholesterol) showed much less nephropathy and mortality than CONV or OAR rats but increased liver fat. Prevention of coprophagy did not change the response of OAR rats to choline deficiency, nor did neomycin. Vitamin B12 and chlortetracycline decreased the nephropathy and mortality of OAR rats but not liver fat. Vitamin B12 and neomycin had no effect on choline-deficient GF rats. All rats had high liver cholesterol; the GF were the highest. The cholesterol levels were unaffected by choline deficiency, but chlortetracycline and neomycin increased liver cholesterol of choline-deficient rats. We ascribe the lessened nephropathy of GF rats fed choline-deficient diets to their lower metabolic rates, absent bacterial utilization of choline and methionine, greater liver choline synthesis, and lessened decrease in renal acetyl choline. We think the abrupt drop in renal acetyl choline in OAR rats fed such diets leads to nephropathy via renal vasospasm and ischemia.

The role of microbial flora in the hepatotoxicity of chlortetracycline in vivo: A study with germfree mice

Studies with germfree mice have revealed that chlortetracycline (CTC) hepatotoxicity does not depend on secondary effects related to modification of the indigenous microflora but that it is the consequence of a direct drug action. Germfree (GF) and conventionalized (CONV) mice, injected intraperitoneally with large doses of CTC, both developed signs of hepatotoxicity: liver weight, total lipids, and lipid fractions (glycerides, cholesterol, phospholipids) increased, while liver water and protein decreased; activity of the liver enzymes (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, glutamic dehydrogenase, urea cleavage enzyme, and arginase) all decreased, even below that of liver protein concentration. In general, the effect of CTC on the GF mouse was more severe than on the CONV: lipids rose higher, while protein fell lower. In each type of mouse, the concentration of liver glycerides increased most. Fasting exacerbated all these effects especially in the CONV mouse. There were no significant differences between the livers of GF and CONV untreated mice in any of the parameters measured, except for total lipids and glycerides, which were lower in the GF liver. Thus, the presence of an indigenous flora appears not only to attenuate the lipogenic response of the liver to CTC but also to influence the liver lipi composition of the untreated mouse. Our results are consistent with the hypothesis that CTC interferes with protein metabolism of the liver in at least two broad ways: by exerting a catabolic or antianabolic affect, in that liver protein, in general, is decreased after CTC; and by interfering with metalloenzyme action by chelation with metal cofactors, leaving those enzymes relatively unaffected which require no metal cofactors.