Deletion of Serum Amyloid A3 Improves High Fat High Sucrose Diet-Induced Adipose Tissue Inflammation and Hyperlipidemia in Female Mice

Serum amyloid A (SAA) increases in response to acute inflammatory stimuli and is modestly and chronically elevated in obesity. SAA3, an inducible form of SAA, is highly expressed in adipose tissue in obese mice where it promotes monocyte chemotaxis, providing a mechanism for the macrophage accumulation that occurs with adipose tissue expansion in obesity. Humans do not express functional SAA3 protein, but instead express SAA1 and SAA2 in hepatic as well as extrahepatic tissues, making it difficult to distinguish between liver and adipose tissue-specific SAA effects. SAA3 does not circulate in plasma, but may exert local effects that impact systemic inflammation. We tested the hypothesis that SAA3 contributes to chronic systemic inflammation and adipose tissue macrophage accumulation in obesity using mice deficient for Saa3 (Saa3 ^−/−). Mice were rendered obese by feeding a pro-inflammatory high fat, high sucrose diet with added cholesterol (HFHSC). Both male and female Saa3 ^−/− mice gained less weight on the HFHSC diet compared to Saa3^+/+ littermate controls, with no differences in body composition or resting metabolism. Female Saa3 ^−/− mice, but not males, had reduced HFHSC diet-induced adipose tissue inflammation and macrophage content. Both male and female Saa3 ^−/− mice had reduced liver Saa1 and Saa2 expression in association with reduced plasma SAA. Additionally, female Saa3 ^−/− mice, but not males, showed improved plasma cholesterol, triglycerides, and lipoprotein profiles, with no changes in glucose metabolism. Taken together, these results suggest that the absence of Saa3 attenuates liver-specific SAA (i.e., SAA1/2) secretion into plasma and blunts weight gain induced by an obesogenic diet. Furthermore, adipose tissue-specific inflammation and macrophage accumulation are attenuated in female Saa3 ^−/− mice, suggesting a novel sexually dimorphic role for this protein. These results also suggest that Saa3 influences liver-specific SAA1/2 expression, and that SAA3 could play a larger role in the acute phase response than previously thought.     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

Oncostatin M Mediates STAT3-Dependent Intestinal Epithelial Restitution via Increased Cell Proliferation,Decreased Apoptosis and Upregulation of SERPIN Family Members

Objective

Oncostatin M (OSM) is produced by activated T cells, monocytes, and dendritic cells and signals through two distinct receptor complexes consisting of gp130 and LIFR (I) or OSMR-β and gp130 (II), respectively. Aim of this study was to analyze the role of OSM in intestinal epithelial cells (IEC) and intestinal inflammation.

Methods

OSM expression and OSM receptor distribution was analyzed by PCR and immunohistochemistry experiments, signal transduction by immunoblotting. Gene expression studies were performed by microarray analysis and RT-PCR. Apoptosis was measured by caspases-3/7 activity. IEC migration and proliferation was studied in wounding and water soluble tetrazolium assays.

Results

The IEC lines Caco-2, DLD-1, SW480, HCT116 and HT-29 express mRNA for the OSM receptor subunits gp130 and OSMR-β, while only HCT116, HT-29 and DLD-1 cells express LIFR mRNA. OSM binding to its receptor complex activates STAT1, STAT3, ERK-1/2, SAPK/JNK-1/2, and Akt. Microarray analysis revealed 79 genes that were significantly up-regulated (adj.-p≤0.05) by OSM in IEC. Most up-regulated genes belong to the functional categories “immunity and defense” (p = 2.1×10⁻⁷), “apoptosis” (p = 3.7×10⁻⁴) and “JAK/STAT cascade” (p = 3.4×10⁻⁶). Members of the SERPIN gene family were among the most strongly up-regulated genes. OSM significantly increased STAT3- and MEK1-dependent IEC cell proliferation (p<0.05) and wound healing (p = 3.9×10⁻⁵). OSM protein expression was increased in colonic biopsies of patients with active inflammatory bowel disease (IBD).

Conclusions

OSM promotes STAT3-dependent intestinal epithelial cell proliferation and wound healing in vitro. Considering the increased OSM expression in colonic biopsy specimens of patients with active IBD, OSM upregulation may modulate a barrier-protective host response in intestinal inflammation. Further in vivo studies are warranted to elucidate the exact role of OSM in intestinal inflammation and the potential of OSM as a drug target in IBD.     

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.     

The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm ^att, sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ^−/−δ^−/−, J_H ^−/−, IgA^−/−, pIgR^−/−). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using ‘L-mice’ which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm ^att from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most “classical” immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.     

Serum amyloid A3 does not contribute to circulating SAA levels

Adipose tissue secretes proteins like serum amyloid A (SAA), which plays important roles in local and systemic inflammation. Circulating SAA levels increase in obese humans, but the roles of adipose-derived SAA and hyperlipidemia in this process are unclear. We took advantage of the difference in the inducible isoforms of SAA secreted by adipose tissue (SAA3) and liver (SAA1 and 2) of mice to evaluate whether adipose tissue contributes to the circulating pool of SAA in obesity and hyperlipidemia. Genetically obese (ob/ob) mice, but not hyperlipidemic mice deficient in apolipoprotein E (Apoe^−/−), had significantly higher circulating levels of SAA than their littermate controls. SAA1/2 mRNA expression in the liver and SAA3 mRNA expression in intra-abdominal fat were significantly higher in obese than thin mice, but they were not affected by hyperlipidemia in Apoe^−/− mice. However, only SAA1/2 and the constitutive form of SAA (SAA4) could be detected in the circulation by mass spectrometric analysis of HDL, the major carrier of circulating SAA. In contrast, SAA3 could be detected in medium from cultured adipocytes. Our findings indicate that the expression of SAA3 in adipose tissue is upregulated by obesity, but it does not contribute to the circulating pool of SAA in mice.     

A brief elevation of serum amyloid A is sufficient to increase atherosclerosis

Serum amyloid A (SAA) has a number of proatherogenic effects including induction of vascular proteoglycans. Chronically elevated SAA was recently shown to increase atherosclerosis in mice. The purpose of this study was to determine whether a brief increase in SAA similarly increased atherosclerosis in a murine model. The recombination activating gene 1-deficient (rag1^−/−) × apolipoprotein E-deficient (apoe^−/−) and apoe^−/− male mice were injected, multiple times or just once respectively, with an adenoviral vector encoding human SAA1 (ad-SAA); the injected mice and controls were maintained on chow for 12–16 weeks. Mice receiving multiple injections of ad-SAA, in which SAA elevation was sustained, had increased atherosclerosis compared with controls. Strikingly, mice receiving only a single injection of ad-SAA, in which SAA was only briefly elevated, also had increased atherosclerosis compared with controls. Using in vitro studies, we demonstrate that SAA treatment leads to increased LDL retention, and that prevention of transforming growth factor beta (TGF-β) signaling prevents SAA-induced increases in LDL retention and SAA-induced increases in vascular biglycan content. We propose that SAA increases atherosclerosis development via induction of TGF-β, increased vascular biglycan content, and increased LDL retention. These data suggest that even short-term inflammation with concomitant increase in SAA may increase the risk of developing CVD.     

Characterisation of Gut Microbiota in Ossabaw and G?ttingen Minipigs as Models of Obesity and Metabolic Syndrome

Background

Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs.

Methods and Findings

The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs’ cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine.

Conclusion

Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.     

Spred-2 Deficiency Exacerbates Lipopolysaccharide-Induced Acute Lung Inflammation in Mice

Background

Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.

Methods

Wild-type (WT) mice and Spred-2^−/− mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2^−/− mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.

Results

LPS-induced acute lung inflammation was significantly exacerbated in Spred-2^−/− mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2^−/− mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.

Conclusions

The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.     

Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota

The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l⁻¹ HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l⁻¹) and long-chain fructooligosaccharides (0.4 g l⁻¹) (PRE) (n=19–21 per group) for 15 days. Piglets (n=7–8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection.     

Intestine-Specific Mttp Deletion Increases the Severity of Experimental Colitis and Leads to Greater Tumor Burden in a Model of Colitis Associated Cancer

Background

Gut derived lipid factors have been implicated in systemic injury and inflammation but the precise pathways involved are unknown. In addition, dietary fat intake and obesity are independent risk factors for the development of colorectal cancer. Here we studied the severity of experimental colitis and the development of colitis associated cancer (CAC) in mice with an inducible block in chylomicron secretion and fat malabsorption, following intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO).

Methodology/Principal Findings

Mttp-IKO mice exhibited more severe injury with ∼90% mortality following dextran sodium sulfate (DSS) induced colitis, compared to <20% in controls. Intestinal permeability was increased in Mttp-IKO mice compared to controls, both at baseline and after DSS administration, in association with increased circulating levels of TNFα. DSS treatment increased colonic mRNA expression of IL-1β and IL-17A as well as inflammasome expression in both genotypes, but the abundance of TNFα was selectively increased in DSS treated Mttp-IKO mice. There was a 2-fold increase in colonic tumor burden in Mttp-IKO mice following azoxymethane/DSS treatment, which was associated with increased colonic inflammation as well as alterations in cytokine expression. To examine the pathways by which alterations in fatty acid abundance might interact with cytokine signaling to regulate colonic epithelial growth, we used primary murine myofibroblasts to demonstrate that palmitate induced expression of amphiregulin and epiregulin and augmented the increase in both of these growth mediators when added to IL-1βor to TNFα.

Conclusions

These studies demonstrate that Mttp-IKO mice, despite absorbing virtually no dietary fat, exhibit augmented fatty acid dependent signaling that in turn exacerbates colonic injury and increases tumor formation.     

Fetal Exposure to Maternal Inflammation Does Not Affect Postnatal Development of Genetically-Driven Ileitis and Colitis

Background: Chronic inflammatory disorders have been increasing in incidence over the past decades following geographical patterns of industrialization. Fetal exposure to maternal inflammation may alter organ functions and the offspring''s disease risk. We studied the development of genetically-driven ileitis and colitis in response to maternal inflammation using mouse models. Methods: Disease susceptible (Tnf ^ΔARE/+ and IL10^−/−) and disease-free (Tnf ^+/+ and IL10^−/+) offspring were raised in inflamed and non-inflamed dams. Ileal, caecal and colonic pathology was evaluated in the offspring at 8 or 12 weeks of age. Ly6G-positive cells in inflamed sections from the distal ileum and distal colon were analysed by immunofluorescence microscopy. Gene expression of pro-inflammatory cytokines was measured in whole tissue specimens by quantitative PCR. Microarray analyses were performed on laser microdissected intestinal epithelium. Caecal bacterial communities were assessed by Illumina sequencing of 16S rRNA amplicons. Results: Disease severity, the number of infiltrated neutrophils as well as Tnf and Il12p40 mRNA expression were independent of maternal inflammation in the offspring of mouse models for ileitis (Tnf ^ΔARE/+) and colitis (IL10^−/−). Although TNF-driven maternal inflammation regulated 2,174 (wild type) and 3,345 (Tnf ^ΔARE/+) genes in the fetal epithelium, prenatal gene expression patterns were completely overwritten after birth. In addition, co-housing experiments revealed no change in phylogenetic diversity of the offspring''s caecal microbiota in response to maternal inflammation. This is independent of the offspring''s genotype before and after the onset of tissue pathology. Conclusions: Disease risk and activity in mouse models of chronic ileitis and colitis was independent of the fetal exposure to maternal inflammation. Likewise, maternal inflammation did not alter the diversity and composition of offspring''s caecal microbiota, clearly demonstrating that changes of the gene expression program in the fetal gut epithelium were not relevant for the development of chronic inflammatory disorders in the gut.     

Effect of Metformin on Metabolic Improvement and Gut Microbiota

Metformin is commonly used as the first line of medication for the treatment of metabolic syndromes, such as obesity and type 2 diabetes (T2D). Recently, metformin-induced changes in the gut microbiota have been reported; however, the relationship between metformin treatment and the gut microbiota remains unclear. In this study, the composition of the gut microbiota was investigated using a mouse model of high-fat-diet (HFD)-induced obesity with and without metformin treatment. As expected, metformin treatment improved markers of metabolic disorders, including serum glucose levels, body weight, and total cholesterol levels. Moreover, Akkermansia muciniphila (12.44% ± 5.26%) and Clostridium cocleatum (0.10% ± 0.09%) abundances increased significantly after metformin treatment of mice on the HFD. The relative abundance of A. muciniphila in the fecal microbiota was also found to increase in brain heart infusion (BHI) medium supplemented with metformin in vitro. In addition to the changes in the microbiota associated with metformin treatment, when other influences were controlled for, a total of 18 KEGG metabolic pathways (including those for sphingolipid and fatty acid metabolism) were significantly upregulated in the gut microbiota during metformin treatment of mice on an HFD. Our results demonstrate that the gut microbiota and their metabolic pathways are influenced by metformin treatment.     

Host adaptive immunity alters gut microbiota

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1^−/− mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1⁻ mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1^−/− mice compared with the wild type. This enrichment was suppressed when Rag1^−/− mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1^−/− mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota''s composition and diversity.     

iNOS-Dependent Increase in Colonic Mucus Thickness in DSS-Colitic Rats

Aim

To investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease.

Methods

Colitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS −/− mice were used.

Results

Colitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (−16±5 µm vs −14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (−33±4 µm vs −10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS−/− mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment.

Conclusion

Both prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.     

Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing

Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species—the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E−9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E−14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356–896 OTUs) was >2-fold higher than in the MI (112–567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that can effectively breakdown a wide variety of complex polysaccharides.     

Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites

Acetate dominated the extracellular pool of volatile fatty acids (VFAs) in the hindgut fluid of Reticulitermes flavipes, Zootermopsis angusticollis, and Incisitermes schwarzi, where it occurred at concentrations of 57.9 to 80.6 mM and accounted for 94 to 98 mol% of all VFAs. Small amounts of C₃ to C₅ VFAs were also observed. Acetate was also the major VFA in hindgut homogenates of Schedorhinotermes lamanianus, Prorhinotermes simplex, Coptotermes formosanus, and Nasutitermes corniger. Estimates of in situ acetogenesis by the hindgut microbiota of R. flavipes (20.2 to 43.3 nmol · termite⁻¹ · h⁻¹) revealed that this activity could support 77 to 100% of the respiratory requirements of the termite (51.6 to 63.6 nmol of O₂ · termite⁻¹ · h⁻¹). This conclusion was buttressed by the demonstration of acetate in R. flavipes hemolymph (at 9.0 to 11.6 mM), but not in feces, and by the ability of termite tissues to readily oxidize acetate to CO₂. About 85% of the acetate produced by the hindgut microbiota was derived from cellulose C; the remainder was derived from hemicellulose C. Selective removal of major groups of microbes from the hindgut of R. flavipes indicated that protozoa were primarily responsible for acetogenesis but that bacteria also functioned in this capacity. H₂ and CH₄ were evolved by R. flavipes (usually about 0.4 nmol · termite⁻¹ · h⁻¹), but these compounds represented a minor fate of electrons derived from wood dissimilation within R. flavipes. A working model is proposed for symbiotic wood polysaccharide degradation in R. flavipes, and the possible roles of individual gut microbes, including CO₂-reducing acetogenic bacteria, are discussed.     

Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia

Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (Gast^−/−) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1β expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. LacZ reporter mice for Sonic hedgehog (Shh), Gli1, and Gli2 expression bred onto the Gast^−/− background revealed reduced Shh and Gli1 expression in the antra compared to wild type controls (WT). Gli2 expression in the Gast^−/− corpus was unchanged. However in the hyperplastic Gast^−/− antra, Gli2 expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that Gli2 is differentially regulated in the hyperplastic Gast^−/− antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1β and Il-11, which promote gastric epithelial proliferation, were increased in the Gast^−/− stomach along with Infγ. To test if inflammation could account for elevated epithelial Gli2 expression in the Gast^−/− antra, the human gastric cell line AGS was treated with IL-1β and was found to increase GLI2 but decrease GLI1 levels. IL-1β also repressed human GAST gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the GAST promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed Gast expression but induced Il-1β gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate Il-1β expression, repress Gast gene expression and increase proliferation, leading to antral hyperplasia.     

Naturally Occurring Stilbenoid TSG Reverses Non-Alcoholic Fatty Liver Diseases via Gut-Liver Axis

The gut-liver axis is largely involved in the development of non-alcoholic fatty liver disease (NAFLD). We investigated whether 2, 3, 5, 4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) could reverse NAFLD induced by a high-fat diet (HFD) and whether it did so via the gut-liver axis. Results showed that TSG could reduce the accumulation of FFA and it did so by reducing the expression of L-FABP and FATP4. TSG regulated gut microbiota balanced and increased the protein expression of ZO-1 and occludin, which could improve the function of the intestinal mucosal barrier and reduce serum LPS content by about 25%. TSG reduced TL4 levels by 56% and NF-κB expression by 23% relative to the NAFLD model group. This suggests that prevention of NAFLD by TSG in HFD-fed rats is mediated by modulation of the gut microbiota and TLR4/NF-κB pathway, which may alleviate chronic low-grade inflammation by reducing the exogenous antigen load on the host.