Prolonged antibiotic use induces intestinal injury in mice that is repaired after removing antibiotic pressure: implications for empiric antibiotic therapy

Metabolic profiling of urine and fecal extracts, histological investigation of intestinal ilea, and fecal metagenomics analyses were used to investigate effects of prolonged antibiotic use in mice. The study provides insight into the effects of extended empiric antibiotic therapy in humans. Mice were administered a broad-spectrum antibiotic for four consecutive days followed by oral gavage with Clostridium butyricum, an opportunistic gram-positive pathogenic bacteria commonly isolated in fecal and blood cultures of necrotizing enterocolitis patients. Metagenomics data indicated loss of bacterial diversity after 4 days on antibiotics that was restored after removing antibiotic pressure. Histological analyses indicated damage to ileal villi after antibiotic treatment that underwent repair after lifting antibiotic pressure. Metabolic profiling confirmed intestinal injury in antibiotic-treated mice indicated by increased urinary trans-4-hydroxy-l-proline, a breakdown product of collagen present in connective tissue of ileal villi that may serve as a biomarker for antibiotic-induced injury in at risk populations.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Prolonged fasting induces long-lasting metabolic consequences in mice

To endure prolonged fasting, animals undergo important acute physiological adjustments. However, whether severe fasting also leads to long-term metabolic adaptations is largely unknown. Forty-eight-hour fasting caused a pronounced weight loss in adult C57BL/6 male mice. Seven days of refeeding increased body adiposity to levels above baseline, whereas fasting-induced reductions in lean body mass and energy expenditure were not fully recovered. Respiratory exchange ratio and locomotor activity also remained altered. A fasting/refeeding cycle led to persistent suppression of Pomc mRNA levels and significant changes in the expression of histone deacetylases and DNA methyltransferases in the hypothalamus. Additionally, histone acetylation in the ventromedial nucleus of the hypothalamus was reduced by prolonged fasting and remained suppressed after refeeding. Mice subjected to 48-h fasting 30 days earlier exhibited higher body weight and fat mass compared to aged-matched animals that were never food-deprived. Furthermore, a previous fasting experience altered the changes in body weight, lean mass, energy expenditure and locomotor activity induced by a second cycle of fasting and refeeding. Notably, when acutely exposed to high-palatable/high-fat diet, mice that went through cumulative fasting episodes presented higher calorie intake and reduced energy expenditure and fat oxidation, compared to mice that had never been subjected to fasting. When chronically exposed to high-fat diet, mice that experienced cumulative fasting episodes showed higher gain of body and fat mass and reduced energy expenditure and calorie intake. In summary, cumulative episodes of prolonged fasting lead to hypothalamic epigenetic changes and long-lasting metabolic adaptations in mice.     

Supplementation with a probiotic mixture accelerates gut microbiome maturation and reduces intestinal inflammation in extremely preterm infants

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Centromeric interval of chromosome 4 derived from C57BL/6 mice accelerates type 1 diabetes in NOD.CD72 congenic mice

The nonobese diabetic (NOD) mouse is a useful model of autoimmune type 1 diabetes exhibiting many similarities to human type 1 diabetes patients including the presence of auto-reactive T cells and pancreas-specific autoantiboies. Multiple Idd loci control the development of diabetes in NOD mice. CD72, a B cell membrane-bound glycoprotein carrying a C-type lectin-like domain, is an inhibitory co-receptor of the B cell antigen receptor (BCR) that negatively regulates BCR signaling. Among four known haplotypes of mouse CD72, NOD mice carry the CD72^c haplotype, whereas most of the other inbred strains of mice carry either CD72^a or CD72^b. In this study, we generated congenic NOD.CD72^b mice that carry C57BL/6 (B6) mouse-derived centromeric chromosome 4 interval (24-45 cM) surrounding the CD72^b locus. Unexpectedly, NOD.CD72^b mice were not protected from diabetes, but rather exhibited accelerated development of both insulitis and diabetes. Our result defines novel locus or loci in the vicinity of CD72 gene that negatively control diabetes, indicating that NOD disease is under complex genetic controls of not only Idd genes but also disease-resistant genes.     

Early induction of diabetes in NOD mice by streptozotocin

To clarify whether the non-obese diabetes prone (NOD) mouse has an unusual pancreatic sensitivity to damage, mice were administered streptozotocin in high dose (direct beta cell toxic) or multiple low-dose (autoimmune-insulitis generating) regimen. NOD mice were found to be less sensitive to the diabetogenic effects of high-dose streptozotocin than C57BL/6 mice, but were exquisitely responsive to the multiple low dose regimen when compared to C57BL/6 or C3H/HeJ mice. These results suggest that the basic defect in NOD mice resides in the immune system and that the NOD mouse may be a useful model to investigate the relationships between environmental factors and intrinsic genetic predisposition to diabetes.     

Targeted disruption of CD38 accelerates autoimmune diabetes in NOD/Lt mice by enhancing autoimmunity in an ADP-ribosyltransferase 2-dependent fashion

Ubiquitously expressed CD38 and T cell-expressed ADP-ribosyltransferase 2 (ART2) are ectoenzymes competing for NAD substrate. CD38 exerts pleiotropic actions in hemopoietic and nonhemopoietic compartments via effects on calcium mobilization. ART2 is an ADP-ribosyltransferase on naive CD4+ and CD8+ T cells. ART2-catalyzed ADP-ribosylation of the P2X7 purinoreceptor elicits apoptosis. Transfer of a genetically disrupted CD38 allele into the autoimmune diabetes-prone NOD/Lt background accelerated diabetes onset in both sexes, whereas transfer of a disrupted ART2 complex had no effect. However, the fact that the accelerated pathogenesis mediated by CD38 deficiency required ART2 activity was demonstrated by combining both ART2 and CD38 deficiencies. Reciprocal bone marrow reconstitution studies demonstrated accelerated diabetes only when CD38-deficient bone marrow was transferred into CD38-deficient recipients. Neither decreases in beta cell function nor viability were indicated. Rather, the balance between T-effectors and T-regulatory cells was disturbed in CD38-deficient but ART2-intact NOD mice. In these mice, significant reductions in total viable CD8+ T cells were observed. This was accompanied by an age-dependent increase in a diabetogenic CD8 clonotype. This in turn correlated with impaired T-regulatory development (10-fold reduction in Foxp3 mRNA expression). These changes were corrected when CD38 deficiency was combined with ART2 deficiency. Both ART2-deficient and CD38/ART2 combined deficient T cells were resistant to NAD-induced killing in vitro, whereas CD38-deficient but ART2-intact T cells showed increased sensitivity, particularly the CD4+ CD25+ subset. Unexpectedly, diabetes development in the combined CD38/ART2 stock was strongly suppressed, possibly through epistatic interactions between genes linked to the targeted CD38 on Chromosome 5 and the ART2 complex on Chromosome 7.     

Immunomodulation of antigen presenting cells promotes natural regulatory T cells that prevent autoimmune diabetes in NOD mice

Progression towards type 1 diabetes (T1D) in susceptible patients is linked to a progressive decline in the capacity of regulatory T cells (Treg) to maintain tolerance. As such, therapies aimed at redressing the failing Treg compartment have been the subject of intense investigation. Treg dysfunction in T1D has recently been linked to a reduced capacity of antigen presenting cells (APCs) to maintain Treg function rather than Treg intrinsic defects. This suggests that therapies aimed simply at addressing the failing Treg compartment are unlikely to provide long-term protection. Here, we demonstrate that modulation of the inflammatory status of CD11b+CD11c- APCs favors the upregulation of protective Tregs in a mouse model of T1D. We further demonstrate that reduced expression of the costimulatory molecule CD40 plays a role in this increased immunoregulatory capacity. Strikingly, Treg upregulation resulted exclusively from an increase in natural Tregs rather than the peripheral conversion of conventional T cells. This suggests that modulation of CD11b+ CD11c- APCs inflammatory properties favors the establishment of natural Treg responses that, unlike adaptive Treg responses, are likely to maintain tolerance to a broad range of antigens. As such, modulation of this APC subset represents a potential therapeutic avenue to reestablish peripheral tolerance and protect from autoimmune diseases such as T1D.     

Effects of plasmid DNA injection on cyclophosphamide-accelerated diabetes in NOD mice

Type 1 diabetes results in most cases from the destruction of insulin-secreting beta cells by the immune system. Several immunization methods based on administration of autoantigenic polypeptides such as insulin and glutamic acid decarboxylase (GAD) have been used to prevent autoimmune diabetes in the non-obese diabetic (NOD) mouse. In the work presented here, a gene-based approach was taken for a similar purpose. A plasmid carrying different cDNAs was used to investigate the effects of injecting naked DNA on cyclophosphamide-accelerated diabetes in female NOD mice. Four-week-old animals received intramuscular injections of plasmid DNA encoding either intracellular GAD, a secreted form of GAD, or a secreted form of a soft coral luciferase. Monitoring of glycosuria and hyperglycemia indicated that injection of plasmid DNA encoding secreted GAD and secreted luciferase could prevent and delay diabetes, respectively. In contrast, injection of DNA encoding intracellular GAD did not suppress the disease significantly. Analysis of anti-GAD IgG(1) antibody titers in animal sera indicated that diabetes prevention after injection of GAD-encoding DNA was possibly associated with increased Th2-type activity. These results suggest that cellular localization of GAD is a factor to consider in the design of GAD-based genetic vaccines for the prevention of autoimmune diabetes.     

Prolonged fasting in mice: a more sensitive approach to genetic diabetes.

Effects of two different periods of fasting were studied on glucose tolerance and insulin response to glucose in genetically diabetic KK and nondiabetic C57BL/6J mice. Blood sugar levels of the KK mice did not differ markedly from those of the C57BL/6J mice at the fed state or after 8 h fasting. They were, however, significantly higher in the KK mice when fasted for 18 h. The serum IRI levels, which were at least twice as high in the KK mice, decreased more markedly after 18 h fasting. The KK mice showed impaired glucose tolerance after 8 h fasting, which became more pronounced after 18 h fasting. The insulin response to glucose in the KK mice was not altered after an 8-hour fast; it was, however, diminished greatly after an 18-hour fast. These data suggest that prolonged fasting is necessary to detect the diabetic traits in the KK mice. The C57BL/6J mice showed neither impaired glucose tolerance nor diminished insulin response to glucose at both periods of fasting. Studies with the F1 hybrids (KK male X C57BL/6J female), which carry half of the diabetic genes, suggest that the mode of inheritance of diabetes in the KK mice might be polygenic.     

Rotavirus infection accelerates type 1 diabetes in mice with established insulitis

Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet beta cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged > or = 12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8(+) T-cell proportions in islets. Levels of beta-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after beta-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of beta cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.     

Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment

Background

The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis result from alterations in intestinal microbes and the immune system. However, the precise dysfunctions of microbial metabolism in the gastrointestinal microbiome during IBD remain unclear. We analyzed the microbiota of intestinal biopsies and stool samples from 231 IBD and healthy subjects by 16S gene pyrosequencing and followed up a subset using shotgun metagenomics. Gene and pathway composition were assessed, based on 16S data from phylogenetically-related reference genomes, and associated using sparse multivariate linear modeling with medications, environmental factors, and IBD status.

Results

Firmicutes and Enterobacteriaceae abundances were associated with disease status as expected, but also with treatment and subject characteristics. Microbial function, though, was more consistently perturbed than composition, with 12% of analyzed pathways changed compared with 2% of genera. We identified major shifts in oxidative stress pathways, as well as decreased carbohydrate metabolism and amino acid biosynthesis in favor of nutrient transport and uptake. The microbiome of ileal Crohn's disease was notable for increases in virulence and secretion pathways.

Conclusions

This inferred functional metagenomic information provides the first insights into community-wide microbial processes and pathways that underpin IBD pathogenesis.     

Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice

Dopamine receptors have five isoforms, termed D1-D5. The D1 and D5 receptors form the D1-like group that couples with the Gαs class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Gαi class of G proteins. In our previous studies, a D1-like-R antagonist, SCH23390, inhibited DC-mediated Th17 differentiation and exhibited preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE) in mice. We herein demonstrate in the current study that in the pancreas obtained from NOD mice, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific markers for Th17. Thereafter, NOD mice were orally administered SCH23390 from week 6 to week 26. At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively (p < 0.05). A histological examination of SCH23390-treated mice exhibited a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islets from vehicle controls showed insulitis. In week 26, spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and exhibited an augmentation of IFNγ induction and the suppression of IL-17 induction in the SCH23390-treated mice. These findings indicate that antagonizing D1-like-R suppresses IL-17 expression, thereby leading to a decreased occurrence of NOD.     

Combination therapy with an antioxidant and a corticosteroid prevents autoimmune diabetes in NOD mice.

Oxygen free radicals have been implicated as mediators of pancreatic islet beta cell damage in autoimmune, insulin-dependent diabetes mellitus (IDDM). In this study, we show that the antioxidant, probucol, produced only a small decrease in diabetes incidence in nonobese diabetic (NOD) mice, an animal model for human IDDM. However, combination of probucol with the antiinflammatory corticosteroid, deflazacort, produced an early synergistic effect, delaying diabetes onset by 3 weeks, and a later additive effect, decreasing diabetes incidence from 68% (17 of 25 mice) to 23% (6 of 26 mice, p < 0.005). Protection against diabetes by the combination of probucol and deflazacort was associated with a significant decrease in pancreatic islet infiltration by macrophages/lymphocytes (insulitis) and prevention of islet beta cell loss.     

Prevention of type I diabetes by low-dose gamma irradiation in NOD mice

Pretreatment with nonlethal, low-dose irradiation has been shown to have a protective effect against oxidative injury in animal tissues. Since oxidative injury of tissues is known to be a major cause of many human diseases, we examined the effect of low-dose irradiation on the progression of type I diabetes in mice. Nonobese diabetic (NOD) mice were treated with gamma irradiation and the progression of the disease was monitored. An elevated level of glucose in urine was first detected at 15 weeks of age in the control NOD mice, whereas the detection was delayed as long as 7 weeks when the mice received a single dose of 0.5 Gy total-body irradiation between 12 and 14 weeks of age. The greatest effect was observed in the mice irradiated at 13 weeks of age. The increase in blood glucose and decrease in blood insulin were effectively suppressed by irradiation at 13 weeks of age. Both suppression of cell death by apoptosis and an increase in superoxide dismutase (SOD) activity were observed in the pancreas 1 week after irradiation. The results indicate that treatment with 0.5 Gy gamma rays suppresses progression of type I diabetes in NOD mice. This is the first report on the preventive effect of low-dose irradiation on disease progression.     

NK cells are not required for spontaneous autoimmune diabetes in NOD mice

NK cells have been shown to either promote or protect from autoimmune diseases. Several studies have examined the role of receptors preferentially expressed by NK cells in the spontaneous disease of NOD mice or the direct role of NK cells in acute induced disease models of diabetes. Yet, the role of NK cells in spontaneous diabetes has not been directly addressed. Here, we used the NOD.NK1.1 congenic mouse model to examine the role of NK cells in spontaneous diabetes. Significant numbers of NK cells were only seen in the pancreas of mice with disease. Pancreatic NK cells displayed an activated surface phenotype and proliferated more than NK cells from other tissues in the diseased mice. Nonetheless, depletion of NK cells had no effect on dendritic cell maturation or T cell proliferation. In spontaneous disease, the deletion of NK cells had no significant impact on disease onset. NK cells were also not required to promote disease induced by adoptively transferred pathogenic CD4(+) T cells. Thus, NK cells are not required for spontaneous autoimmune diabetes in NOD mice.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

A wheat-based,diabetes-promoting diet induces a Th1-type cytokine bias in the gut of NOD mice

Dietary antigens are candidate environmental factors in the pathogenesis of type 1 diabetes. In the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, cereal-based diets promote disease development, whereas the diets based on hydrolysed proteins or non-diabetogenic proteins are protective. The hypothesis that diabetogenic diets modulate the cytokine balance in the gut was tested. NOD mice were fed with NTP-2000 (mainly a wheat-based milk-free diet) or Prosobee (a semi-purified hypoallergenic diet based on soy protein isolate) or Prosobee plus casein (milk protein fraction). The mRNA levels of IFN-gamma, IL-10, TNF-alpha, TGF-beta, and inducible NO synthase in the small intestine and the Peyer's patches were determined by semi-quantitative RT-PCR. Mice fed on the cereal-based NTP-2000 diet expressed higher levels of the Th1-type and pro-inflammatory markers IFN-gamma, TNF-alpha, and inducible NO synthase mRNA compared to the Prosobee-fed animals. The expression of the counterregulatory cytokines IL-10 and TGF-beta was unaffected. This resulted in a significant bias of the intestinal cytokine balance towards T helper cell type 1 after feeding NTP-2000. The cytokine mRNA levels in the gut-associated Peyer's patches were not affected. Thus, modulation of gut immunoreactivity by diet may contribute to disease development in NOD mice.