Microbiota Reconstitution Does Not Cause Bone Loss in Germ-Free Mice

ABSTRACTAnnually, an estimated 2 million osteoporotic fractures occur in the United States alone. Osteoporosis imparts a great burden on the health care system. The identification of novel regulators of bone health is critical for developing more effective therapeutics. A previous study on the colonization of germ-free (GF) mice with a microbial community has demonstrated that bacterial colonization dramatically increases bone loss. We therefore investigated the impact of multiple microbial communities in different mice to understand how generalizable the impact of bacterial colonization is on bone health. To investigate the impact of different microbial communities on bone health in outbred and inbred mouse strains, gavage was performed on GF Swiss Webster and GF C57BL/6 mice to introduce distinct microbiotas that originated from either humans or mice. GF mice displayed a high degree of colonization, as indicated by more than 90% of the operational taxonomic units present in the starting inoculum being successfully colonized in the mice when they were examined at the end of the experiment. In spite of the successful colonization of GF mice with gut microbiota of either mouse or human origin, bone mass did not change significantly in any of the groups tested. Furthermore, static and dynamic bone parameters and osteoclast precursor and T cell populations, as well as the expression of several inflammatory markers, were mostly unchanged following microbial colonization of GF mice.IMPORTANCEThe microbiota has been shown to be an important regulator of health and development. With regard to its effect on bone health, a previous study has suggested that gut microbes negatively impact bone density. However, we show here that this is not generalizable to all microbial communities and mouse strain backgrounds. Our results demonstrate that colonization of mice, both outbred and inbred strains, did not have a major impact on bone health. The identification of microbial communities that do not negatively impact bone health may provide a foundation for future investigations that seek to identify microbes that are either beneficial or detrimental to bone metabolism.

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Genetic background influences the impact of KLOTHO deficiency

Physiological Genomics ◽

10.1152/physiolgenomics.00094.2020 ◽

2020 ◽

Vol 52 (10) ◽

pp. 512-516

Author(s):

Jawad S. Salloum ◽

Diane E. Garsetti ◽

Melissa B. Rogers

Keyword(s):

Genetic Background ◽

Genetic Architecture ◽

Disease Susceptibility ◽

Inbred Mouse ◽

Mouse Strains ◽

Disease Models ◽

Inbred Mouse Strains ◽

Metabolic Interactions ◽

Klotho Protein ◽

The Impact

Genetic background is a key but sometimes overlooked factor that profoundly impacts disease susceptibility and presentation in both humans and disease models. Here we show that deficiency of KLOTHO protein, an important renal regulator of mineral homeostasis and a cofactor for FGF23, causes different phenotypes in 129S1/SvlmJ (129) and C57BL/6J (B6) mouse strains. The 129 strain is more severely affected, with decreased longevity, decreased body weight, and increased amounts of kidney calcification compared with B6 mice. Reciprocal F1 crosses of the strains also indicate a parentage effect on the Klotho phenotype with F1 KLOTHO-deficient progeny of B6 mothers and 129 fathers having more kidney calcification than progeny of 129 mothers and B6 fathers. Comparing and contrasting the genetic architecture leading to different phenotypes associated with specific inbred mouse strains may reveal previously unrecognized and important metabolic interactions affecting chronic kidney disease.

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Sickle cell disease promotes sex-dependent pathological bone loss through enhanced cathepsin proteolytic activity

Blood Advances ◽

10.1182/bloodadvances.2021004615 ◽

2021 ◽

Author(s):

Jada M Selma ◽

Hannah Song ◽

Christian Rivera ◽

Simone Andrea Douglas ◽

Abhiramgopal Akella ◽

...

Keyword(s):

Sickle Cell Disease ◽

Bone Loss ◽

Sickle Cell ◽

Sickle Cell Trait ◽

Bone Microarchitecture ◽

The United States ◽

Cell Disease ◽

Female Mice ◽

Blood Disorder ◽

The Impact

Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis and osteopenia and other bone related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD)1,2. Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to study the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by high-resolution quantitative micro-computed tomography (microCT) and the 3D reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. Particularly, SCD female mice are prone to trabecular bone loss while cortical bone degradation occurs in both sexes. Additionally, we describe the impact of genetic knockdown of cathepsin K and E-64 mediated cathepsin inhibition on SBD.

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Microbial Colonization in Adulthood Shapes the Intestinal Macrophage Compartment

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz036 ◽

2019 ◽

Vol 13 (9) ◽

pp. 1173-1185 ◽

Cited By ~ 2

Author(s):

Franziska Schmidt ◽

Katja Dahlke ◽

Arvind Batra ◽

Jacqueline Keye ◽

Hao Wu ◽

...

Keyword(s):

Intestinal Wall ◽

Functional Restoration ◽

Microbial Colonization ◽

Germ Free ◽

Adult Mice ◽

Immune Mediated ◽

Complete Restoration ◽

Specific Pathogen ◽

Intestinal Macrophage ◽

The Impact

Abstract Background and Aims Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. Methods Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. Results Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. Conclusions This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.

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A231 THE MICROBIOTA-NOCICEPTORS-MICROGLIA AXIS CONTROLS THE DEVELOPMENT AND MAINTENANCE OF CHRONIC VISCERAL HYPERSENSITIVITY

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwab002.229 ◽

2021 ◽

Vol 4 (Supplement_1) ◽

pp. 279-280

Author(s):

N Abdullah ◽

C Altier

Keyword(s):

Pain Threshold ◽

Bacterial Colonization ◽

Commensal Bacteria ◽

Phenotypic Characterization ◽

Visceral Sensitivity ◽

Nociceptive Neurons ◽

Germ Free ◽

Thermal Pain ◽

Hyperalgesic Priming ◽

The Impact

Abstract Background Pain is the most common cause of disability in inflammatory bowel disease (IBD). Current medical interventions control the debilitating clinical symptoms by reducing gastrointestinal (GI) inflammation. Despite successful treatment of active disease, abdominal pain persists during remission, suggesting a high level of plasticity in pain-sensing circuits (hyperalgesic priming) caused by inflammation. What drives this remodelling has remained elusive. We have identified microglia as active players of hyperalgesic priming in IBD. Furthermore, it was recently shown that commensal bacteria control the maturation of microglia in the CNS, suggesting that dysbiosis could influence visceral sensitivity through regulating colonic nociceptors-microglia interaction. Here we test the hypothesis that microbiome-nociceptors-microglia interactions control visceral sensitivity and pain in IBD. Aims We investigated the role of the microbiota in the developmental regulation of colonic nociceptors that express the pain receptor TRPV1. We will identify the microbial factors that control neuron-microglia interactions during bacterial colonization and post-inflammatory dysbiosis. Methods We have developed a germ-free TRPV1-GFP reporter mouse to be used for a combination of behavioural tests and phenotypic characterization of TRPV1+ nociceptors. RNA-sequencing of FACS isolated TRPV1+ neurons of germ-free mice will be used to identify genes that are under the control of the microbiota. We will restore discrepancies observed in germ-free mice by recolonization to assess the impact of the microbiota. Furthermore, we will investigate the regulation of Ahr in TRPV1+ neurons by the microbiota and the effect of its ligands on microglial activation and post-inflammatory visceral pain. Results Measuring somatic pain sensation in naive germ-free and SPF mice, we showed a 15% reduction in thermal pain threshold, as measured by the Hargreaves test, and a 50% reduction in mechanical pain threshold, as measured by the Von Frey test, in germ-free mice. When looking at the dorsal root ganglia of germ-free and SPF mice, we saw a 15% increase in the percentage of neurons that were TRPV1-GFP positive in germ-free mice. Conclusions Our results thus far highlight the importance of the microbiota in regulating the lineage of nociceptive neurons and the threshold of mechanical and thermal pain responses. These findings suggest a major contribution of the microbiota in shaping the neuro-immune axis, with major implications for visceral sensitization in the context of dysbiosis. My project will be looking further into the phenotype of nociceptors in germ-free mice and the effect of microbial-derived Ahr agonists on the maturation and function of colonic TRPV1+ nociceptors. My work will advance our understanding of mechanisms by which commensal bacteria regulate GI pain. Funding Agencies CIHR

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Genetic Variability affects the Skeletal Response to Unloading

10.1101/2020.06.26.174326 ◽

2020 ◽

Author(s):

Michael A. Friedman ◽

Abdullah Abood ◽

Bhavya Senwar ◽

Yue Zhang ◽

Camilla Reina Maroni ◽

...

Keyword(s):

Bone Loss ◽

Genetic Variability ◽

Inbred Mouse ◽

Bone Volume ◽

Mouse Strains ◽

Mineral Density ◽

Mechanical Unloading ◽

Bone Response ◽

Trabecular Bone Loss ◽

Resolution Mapping

AbstractMechanical unloading decreases bone volume and strength. In humans and mice, bone mineral density is highly heritable, and in mice the response to changes in loading varies with genetic background. Thus, genetic variability may affect the response of bone to unloading. As a first step to identify genes involved in bone response to unloading, we evaluated the effects of unloading in eight inbred mouse strains: C57BL/6J, PWK/PhJ, WSB/EiJ, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, and CAST/EiJ. C57BL/6J and NOD/ShiLtJ mice had the greatest unloading induced loss of diaphyseal cortical bone volume and strength. NZO/HlLtJ mice had the greatest metaphyseal trabecular bone loss, and C57BL/6J, WSB/EiJ, NOD/ShiLtJ, and CAST/EiJ mice had the greatest epiphyseal trabecular loss. Bone loss in the epiphyses displayed the highest heritability. With immobilization, mineral:matrix was reduced, and carbonate:phosphate and crystallinity were increased. A/J mice displayed the greatest unloading induced loss of mineral:matrix. Changes in gene expression in response to unloading were greatest in NOD/ShiLtJ and CAST/EiJ mice. The most upregulated genes in response to unloading were associated with increased collagen synthesis and extracellular matrix formation. Our results demonstrate a strong differential response to unloading as a function of strain. Diversity outbred (DO) mice are a high-resolution mapping population derived from these eight inbred founder strains. These results suggest DO mice will be highly suited for examining the genetic basis of the skeletal response to unloading.

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Modeling the Genetic Basis of Individual Differences in Susceptibility to Gulf War Illness

Brain Sciences ◽

10.3390/brainsci10030143 ◽

2020 ◽

Vol 10 (3) ◽

pp. 143 ◽

Cited By ~ 2

Author(s):

Byron C. Jones ◽

Diane B. Miller ◽

Lu Lu ◽

Wenyuan Zhao ◽

David G. Ashbrook ◽

...

Keyword(s):

Individual Differences ◽

Inbred Mouse ◽

Combined Treatment ◽

Reference Population ◽

Gulf War ◽

Cytokine Gene Expression ◽

Mouse Strains ◽

Genetic Constitution ◽

Gulf War Illness ◽

The Impact

Between 25% and 30% of the nearly one million military personnel who participated in the 1991 Persian Gulf War became ill with chronic symptoms ranging from gastrointestinal to nervous system dysfunction. This disorder is now referred to as Gulf War Illness (GWI) and the underlying pathophysiology has been linked to exposure-based neuroinflammation caused by organophosphorous (OP) compounds coupled with high circulating glucocorticoids. In a mouse model of GWI we developed, corticosterone was shown to act synergistically with an OP (diisopropylflurophosphate) to dramatically increase proinflammatory cytokine gene expression in the brain. Because not all Gulf War participants became sick, the question arises as to whether differential genetic constitution might underlie individual differences in susceptibility. To address this question of genetic liability, we tested the impact of OP and glucocorticoid exposure in a genetic reference population of 30 inbred mouse strains. We also studied both sexes. The results showed wide differences among strains and overall that females were less sensitive to the combined treatment than males. Furthermore, we identified one OP-glucocorticoid locus and nominated a candidate gene—Spon1—that may underlie the marked differences in response.

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Switchgrass ecotypes alter microbial contribution to deep-soil C

SOIL ◽

10.5194/soil-2-185-2016 ◽

2016 ◽

Vol 2 (2) ◽

pp. 185-197 ◽

Cited By ~ 7

Author(s):

Damaris Roosendaal ◽

Catherine E. Stewart ◽

Karolien Denef ◽

Ronald F. Follett ◽

Elizabeth Pruessner ◽

...

Keyword(s):

Microbial Communities ◽

Relative Abundance ◽

Root Architecture ◽

Microbial Community Composition ◽

Specific Root Length ◽

The United States ◽

Stable Isotope Probing ◽

Gram Negative Bacteria ◽

Gram Negative ◽

The Impact

Abstract. Switchgrass (Panicum virgatum L.) is a C4, perennial grass that is being developed as a bioenergy crop for the United States. While aboveground biomass production is well documented for switchgrass ecotypes (lowland, upland), little is known about the impact of plant belowground productivity on microbial communities down deep in the soil profiles. Microbial dynamics in deeper soils are likely to exert considerable control on ecosystem services, including C and nutrient cycles, due to their involvement in such processes as soil formation and ecosystem biogeochemistry. Differences in root biomass and rooting characteristics of switchgrass ecotypes could lead to distinct differences in belowground microbial biomass and microbial community composition. We quantified root abundance and root architecture and the associated microbial abundance, composition, and rhizodeposit C uptake for two switchgrass ecotypes using stable-isotope probing of microbial phospholipid fatty acids (PLFAs) after 13CO2 pulse–chase labeling. Kanlow, a lowland ecotype with thicker roots, had greater plant biomass above- and belowground (g m−2), greater root mass density (mg cm−3), and lower specific root length (m g−1) compared to Summer, an upland ecotype with finer root architecture. The relative abundance of bacterial biomarkers dominated microbial PLFA profiles for soils under both Kanlow and Summer (55.4 and 53.5 %, respectively; P  =  0.0367), with differences attributable to a greater relative abundance of Gram-negative bacteria in soils under Kanlow (18.1 %) compared to soils under Summer (16.3 %; P  =  0.0455). The two ecotypes also had distinctly different microbial communities process rhizodeposit C: greater relative atom % 13C excess in Gram-negative bacteria (44.1 ± 2.3 %) under the thicker roots of Kanlow and greater relative atom % 13C excess in saprotrophic fungi under the thinner roots of Summer (48.5 ± 2.2 %). For bioenergy production systems, variation between switchgrass ecotypes could alter microbial communities and impact C sequestration and storage as well as potentially other belowground processes.

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The Effect of Population Structure on Murine Genome-Wide Association Studies

10.1101/2020.09.01.278762 ◽

2020 ◽

Author(s):

Meiyue Wang ◽

Gary Peltz

Keyword(s):

Population Structure ◽

Association Studies ◽

Inbred Mouse ◽

Genome Wide Association ◽

Mouse Strains ◽

Genome Wide Association Studies ◽

Inbred Mouse Strains ◽

Minimal Impact ◽

Genome Wide ◽

The Impact

AbstractPopulation structure (PS) has been shown to cause false positive signals in genome-wide association studies (GWAS). Since PS correction is routinely used in human GWAS, it was assumed that it should be utilized for murine GWAS. Nevertheless, there are fundamental differences between murine and human GWAS, and the impact of PS on murine GWAS results has not been thoroughly investigated. We examined 8223 datasets characterizing biomedical responses in panels of inbred mouse strains to assess the impact of PS on murine GWAS. Surprisingly, we found that PS had a minimal impact on datasets characterizing responses in ≤20 strains; and relatively little impact on the majority of datasets characterizing >20 strains. Moreover, there were examples where association signals within known causative genes could be rejected if PS correction methods were utilized. PS assessment should be carefully used, and considered in conjunction with other criteria, for assessing the candidate genes that are identified in murine GWAS.

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The Epiallelic Nature of Mouse rDNA

10.1101/2021.06.10.447887 ◽

2021 ◽

Author(s):

Fran Rodriguez-Algarra ◽

Amy Danson ◽

Rob Seaborne ◽

Selin Ylidizoglu ◽

Haru Yoshikawa ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Variation ◽

Inbred Mouse ◽

Mouse Strains ◽

Epigenetic State ◽

In Utero Environment ◽

Transcriptional Output ◽

The Impact ◽

Human And Mouse ◽

Rdna Copy

Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. Here we report that 45S rDNA units in the C57BL/6J mouse strain are epiallelic, existing as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. Epigenetic dynamics at these haplotypes are dichotomous and lifestage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during ageing only. rDNA epiallelism is influenced by total rDNA copy number, and also found in other inbred mouse strains and humans. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

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Feeding Mode, but Not Prebiotics, Affects Colonic Microbiota Composition and Volatile Fatty Acid Concentrations in Sow-Reared, Formula-Fed, and Combination-Fed Piglets

Journal of Nutrition ◽

10.1093/jn/nxz183 ◽

2019 ◽

Vol 149 (12) ◽

pp. 2156-2163

Author(s):

Mei Wang ◽

Emily C Radlowski ◽

Min Li ◽

Marcia H Monaco ◽

Sharon M Donovan

Keyword(s):

Fatty Acid ◽

Volatile Fatty Acid ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Colonization ◽

Formula Feeding ◽

Microbial Colonization ◽

Large White ◽

Feeding Mode ◽

Bacterial Composition ◽

The Impact

ABSTRACT Background Many infants consume both human milk and infant formula (combination-fed); however, little is known about how combination-feeding affects the gut microbiota or prebiotic fermentation compared to formula feeding. Objectives We investigated the impact of feeding mode and prebiotics on bacterial colonization and volatile fatty acid (VFA) concentrations. Methods Newborn piglets (Large White and Landrace) were randomly assigned to 5 groups (n = 6/group): formula-fed (FF), formula-fed with prebiotics (FP), sow-reared (SR), combination-fed (CF), and combination-fed with prebiotics (CP). SR piglets remained with the sows 24 h/d. FF and FP were fed formula or formula with galactooligosaccharide and inulin (4 g/L in a 4:1 ratio). CF and CP were sow-reared for 5 d and then rotated between the sow and formula-feeding every 12 h. Ascending colon contents were collected at day 21. The microbiota was analyzed by pyrosequencing and denaturing gradient gel electrophoresis (DGGE). VFAs were determined by gas chromatography. Results Distance-based redundancy analysis of DGGE and pyrosequencing data separated microbiota of FF from CF and SR. CF differed from SR by DGGE, but only a trend (P = 0.09) by pyrosequencing. Bacterial composition of CF was more similar to SR than FF. No bacterial genera in CF significantly differed from SR; however, 9 genera differed between CF and FF, including Lactobacillus, Clostridium XIVa, and Fusobacterium. VFA concentrations were similar between CF and SR, while isovalerate and isobutyrate were 2-fold greater (P < 0.05) in CF than FF. Neither microbiota nor VFA profile was affected by prebiotic supplementation. Conclusions Microbial colonization patterns and VFA profiles of CF piglets were more similar to SR piglets than FF piglets. Prebiotics did not affect piglet bacterial composition and/or VFA concentrations relative to the main feeding modes (FF and CF). Thus, partial exposure to breast milk can be beneficial for microbiota development of FF neonates.

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