Specific Pathogen-free Mice Research Articles

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, allpublicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria,and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

Evaluation of exhaust air dust polymerase chain reaction as a supplement method for soiled bedding sentinel monitoring in specific pathogen free mouse facility using two different individually ventilated cage racks.

Health monitoring is essential for ensuring animal health and reliable research results. Each animal facility should establish adequate health monitoring methods, and microbiological quality control should be implemented through regular health surveillance. Recently, specific pathogen free (SPF) mice have been housed in individually ventilated cage (IVC) racks in the majority of mouse facilities globally, and health monitoring is implemented using a soiled bedding sentinel (SBS). Even though SBS monitoring is a standard method, it has a limitation in that some pathogens are not sufficiently transmitted to the sentinel housed in the IVC. The exhaust air dust polymerase chain reaction (EAD PCR) method has been reported to be a reliable complementary method to SBS monitoring based on research findings. In Korea, health monitoring programs using EAD PCR have not yet been applied to laboratory animal facilities. The microbiological status of mouse colonies housed in the two IVC racks was compared using SBS and EAD PCR monitoring in our SPF mouse facility. Except for Helicobacter spp. and Staphylococcus aureus, the detection of 16 pathogens did not differ between the two methods. In the detection of Helicobacter spp., EAD PCR was found to be more sensitive than SBS. Helicobacter spp. were not found by SBS, whereas four S. aureus positive samples were detected by either SBS or EAD PCR test. According to our findings, EAD PCR can be used as a supplement to SBS monitoring. Moreover, EAD PCR can reduce the number of animals used, making it a 3R (Replacement, Reduction, Refinement)-consistent method.

Antitumor Effect and Pharmacological Mechanism of Paclitaxel-loaded Silk Fibroin Nanomaterials on H22 Subcutaneous Tumor Model of Mouse Hepatoma

This work was developed to analyze the adoption of paclitaxel-loaded silk fibroin nanoparticles (SFNPs) in subcutaneous transplanted tumor model of mouse hepatoma to explore its anti-tumor effect. Twenty-five specific pathogen-free (SPF) mice were selected to construct a subcutaneous tumor model of liver cancer, and they were randomly rolled into control group, group A, group B, group C, and group D, with five mice in each group. The silk fibroin was mixed with an organic solvent to prepare a suspension, and the SFNPs were prepared through centrifugation, ultrasound, and other methods. The paclitaxel-loaded SFNPs were prepared by mixing paclitaxel and silk fibroin aqueous solution, centrifuging, washing, and dispersing. Then, the five groups of mice were intervened by different dosage regimens to analyze the changes of various indicators. As a result, the prepared nanoparticles had uniform particle size, uniform distribution, no adhesion, and the average particle size was less than 500 nm. The tumor volume of mice in groups C and D on the 7th, 9th, and 13th days of administration were dramatically smaller than those in the control group, group A, and group B (P [ 0.05). And the tumor volume (154.49 � 9.65 mm3) of mice in group D on the 13th day of administration was dramatically smaller than that in group C (167.79 � 9.72 mm3) (P [ 0.05). The tumor mass (0.89 � 0.14 g, 0.54 � 0.13 g, and 0.46 � 0.11 g) of mice in groups B, C, and D was dramatically smaller than that in the control group and group A (1.23 � 0.12 g, 1.24 � 0.11 g) (P [ 0.05), and that of group D was dramatically smaller than groups B and C (P [ 0.05). The apoptosis rates of tumor cells in groups C and D (46.38%, 48.23%) were greatly superior to those in the control group (16.7%), group A (21.33%), and group B (35.6%) (P [ 0.05), and that of group D was greatly superior to that in group C (P [ 0.05). In summary, paclitaxel-loaded SFNPs can effectively improve the targeting effect and bioavailability of drugs in the treatment of liver cancer, thereby improving the efficacy, and had a good application prospect.

Effect of DSS-Induced Ulcerative Colitis and Butyrate on the Cytochrome P450 2A5: Contribution of the Microbiome.

Several studies have indicated the beneficial anti-inflammatory effect of butyrate in inflammatory bowel disease (IBD) therapy implying attempts to increase butyrate production in the gut through orally administered dietary supplementation. Through the gut–liver axis, however, butyrate may reach directly the liver and influence the drug-metabolizing ability of hepatic enzymes, and, indirectly, also the outcome of applied pharmacotherapy. The focus of our study was on the liver microsomal cytochrome P450 (CYP) 2A5, which is a mouse orthologue of human CYP2A6 responsible for metabolism of metronidazole, an antibiotic used to treat IBD. Our findings revealed that specific pathogen-free (SPF) and germ-free (GF) mice with dextran sulfate sodium (DSS)-induced colitis varied markedly in enzyme activity of CYP2A and responded differently to butyrate pre-treatment. A significant decrease (to 50%) of the CYP2A activity was observed in SPF mice with colitis; however, an administration of butyrate prior to DSS reversed this inhibition effect. This phenomenon was not observed in GF mice. The results highlight an important role of gut microbiota in the regulation of CYP2A under inflammatory conditions. Due to the role of CYP2A in metronidazole metabolism, this phenomenon may have an impact on the IBD therapy. Butyrate administration, hence, brings promising therapeutic potential for improving symptoms of gut inflammation; however, possible interactions with drug metabolism need to be further studied.

Efficacy of Bacillus coagulans BC01 on loperamide hydrochloride-induced constipation model in Kunming mice.

In this study, the laxative effect of Bacillus coagulans BC01 (BC01) in mice was investigated using a functional constipation mouse model. Six-week-old male specific pathogen-free (SPF) Kunming mice were randomly divided into five groups: normal control group (saline), model group (loperamide hydrochloride), drug control group (bisacodyl), BC01 low-dose group (4.0 × 108 CFU/mL) and BC01 high-dose group (4.0 × 109 CFU/mL). Except for the normal group, the functional constipation model was established by administering 0.25 mL of a loperamide hydrochloride suspension (1 mg/mL) twice daily for four consecutive days by oral gavage. After modeling, the BC01 groups were administered 0.25 mL of BC01. The bisacodyl served as a control and was administered orally at a dose of 100 mg/kg, while the other groups were administered 0.25 mL of sterile saline. After 7 days of continuous administration, the experimental mice were again induced by loperamide hydrochloride. During this period, the mechanism of BC01 to improve constipation symptoms in mice was analyzed by measuring the changes in body weight, fecal water content, small intestine propulsion rate, histology of small intestinal tissue sections, fecal microbial diversity, serum indices, as well as mRNA and protein expression levels in the small intestinal tissue. BC01 was found to significantly promote the intestinal propulsion rate and increase the fecal water content in the mice. BC01 could also alleviates constipation by regulating gastrointestinal motility (substance P, motilin, endothelin-1, somatostatin, and vasoactive intestinal peptide), gene expression (c-Kit, SCF, COX-2, NF-κB, iNOS, and eNOS), intestinal inflammation (eNOS, iNOS, NF-κB), and the intestinal microbiota composition in the constipated mice. In addition, the high-dose BC01 treatment had the best preventive effect on constipation. BC01 is a probiotic strain to effectively relieve the adverse effects of constipation.

Multi-omics analysis reveals the effects of microbiota on oral homeostasis.

The oral epithelium’s normal morphological structure and function play an important role in maintaining oral homeostasis, among which microbiota and chronic stress are key contributing factors. However, the effects of microbiota and chronic stress on the morphological structures and molecular function of oral homeostasis remain unclear. In this study, morphological staining was used to compare the tongue structure of specific pathogen-free and germ-free mice, and an integrated multi-omics analysis based on transcriptomics, proteomics, and metabolomics was performed to investigate the regulatory mechanisms of microbiota and chronic stress on oral homeostasis. We found that the morphological structure of the tongue in germ-free mice was disordered compared with in specific pathogen-free mice, especially in the epithelium. Multi-omics analysis indicated that differentially expressed molecules of the tongue between germ-free and specific pathogen-free mice were significantly enriched in the mitochondrial metabolic process and immune response. Interestingly, microbiota also significantly influenced the permeability of the oral epithelial barrier, represented by the differential expression of keratinization, and cell adhesion molecules. It was worth noting that the above changes in the tongue between specific pathogen-free and germ-free mice were more significant after chronic stress. Collectively, this is the first study to reveal that the microbiota might maintain oral homeostasis by reshaping the structure of the oral epithelial barrier and changing the function of molecular biology, a process that may be driven by the immune response and mitochondrial metabolic process of oral tissue. Furthermore, chronic stress can enhance the regulatory effects of microbiota on oral homeostasis.

Absence of Gut Microbiota Is Associated with RPE/Choroid Transcriptomic Changes Related to Age-Related Macular Degeneration Pathobiology and Decreased Choroidal Neovascularization.

Studies have begun to reveal significant connections between the gut microbiome and various retinal diseases, including age-related macular degeneration (AMD). As critical supporting tissues of the retina, the retinal pigment epithelium (RPE) and underlying choroid play a critical role in retinal homeostasis and degeneration. However, the relationship between the microbiome and RPE/choroid remains poorly understood, particularly in animal models of AMD. In order to better elucidate this role, we performed high-throughput RNA sequencing of RPE/choroid tissue in germ-free (GF) and specific pathogen-free (SPF) mice. Furthermore, utilizing a specialized laser-induced choroidal neovascularization (CNV) model that we developed, we compared CNV size and inflammatory response between GF and SPF mice. After correction of raw data, 660 differentially expressed genes (DEGs) were identified, including those involved in angiogenesis regulation, scavenger and cytokine receptor activity, and inflammatory response—all of which have been implicated in AMD pathogenesis. Among lasered mice, the GF group showed significantly decreased CNV lesion size and microglial infiltration around CNV compared to the SPF group. Together, these findings provide evidence for a potential gut–RPE/choroidal axis as well as a correlation with neovascular features of AMD.

Wnt 5a mediated inflammatory injury of renal tubular epithelial cells dependent on calcium signaling pathway in Trichloroethylene sensitized mice

Patients with trichloroethene-induced Trichloroethylene hypersensitivity syndrome (THS) often present kidney injury. However, the role of Wnt 5a/Ca2+ pathway in renal tubular injury in Trichloroethylene (TCE) sensitized mice remains unclear. This study aimed to investigate how Wnt 5a/Ca2+ pathway induced renal tubular epithelial cell injury in TCE sensitized mice. A total of 84 female BALB/c Specific Pathogen Free mice aged 6–8 weeks were used to establish TCE sensitized mouse models. Renal histology and serum levels of α1-MG and β2-MG were used to assess the renal injury. The renal protein levels of Wnt 5a, ROR2, FZD5, PLC, p-CaMKII, IκB α, p-IκB α, NF-κB(p65), TNF α, IL 6 and IL 1β were measured. The levels of serum α1-MG and β2-MG and TNF α, IL 6 and IL 1β levels in the kidney tissue were significantly increased in TCE sensitized positive group. However, Box5 pretreatment inhibited the expression of PLC, p-CaMKII, p65 and attenuated the injury of renal tubular epithelial cells and suppressed the upregulated expression of the above cytokines. In addition, KN93 also reduced nuclear translocation of p65 and renal injury as well as the elevated cytokines by inhibiting CaMKII. These data identify Wnt 5a binding to ROR2 and FZD5, p65 nuclear translocation, and inflammatory cytokine release as a novel mechanism for renal tubular epithelial cells injury by sensitization with TCE. Box5 or KN93 pretreatment can block the expression of inflammatory cytokines and reduce the injury of renal tubular epithelial cells.

563 Commensal microbes can regulate skin barrier through the control of tryptophan-aryl hydrocarbon receptor signaling cascade

Commensal microbes are critical in maintaining skin homeostasis. However, their mechanisms of crosstalk with host epithelia during barrier disruption and repair are not defined. Using germ free (GF) mice, we have recently demonstrated that microbiota is necessary and sufficient for proper differentiation and repair of the epidermal barrier. By comparing epithelial transcriptomes of GF mice to specific pathogen free (SPF) mice we found that microbiota regulate genes involved in epithelial development and differentiation.

Diarrheal Mice Induced by a High-fat and High-protein Diet could be Cured by Bohe Pill Regulating Intestinal Mucosal Bacteria

This study aimed to investigate the effect of Baohe pill decoction on the bacterial diversity and community structure of the intestinal mucosa of mice with diarrhea caused by feeding HFD and HPD, and provides a scientific basis for the efficacy of Baohe pill decoction in treating this condition. Specific pathogen-free Kunming male mice were randomized into three equal groups, i.e. the control (ftcm), natural recovery (ftmm), and Baohe pill decoction treatment (fttm) groups. The ftcm group was fed a general feed diet, and the ftmm and fttm groups were treated with HFD and HPD. Following the successful induction of diarrhea, the mice in the fttm group were gavaged with Baohe pill decoction, and the mice in the ftmm group were given equal amounts of sterile water. The intestinal mucosal microbiota characteristics were analyzed by 16S ribosomal ribonucleic acid Pacbio single-molecule, real-time full-length gene sequencing. The operational taxonomic unit (OTU) number and alpha diversity in the intestinal mucosal flora of the fttm group were higher than in the ftcm and ftmm groups (P > 0.05), and the composition of the intestinal mucosa bacteria differed between the ftmm and fttm groups. The ftmm group showed a higher abundance of Proteobacteria, Actinobacteria, and Streptophyta, while the Firmicutes, Bacteroidetes, and Verrucomicrobia in the fttm group were higher than that in the other two groups. Compared with the ftmm group, certain beneficial bacteria (Lactobacillus) showed an increased abundance, and pathogenic bacteria (Bacillus, Bradyrhizobium, and Ralstonia) showed a decreased abundance. Bacterial species, such as Lactobacillus (Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus acidophilus and Lactobacillus intestinalis) were significantly higher in the fttm and closely matched those in the ftcm group. Furthermore, various opportunistic pathogens caused by feeding an HFD and HPD were not detected in the fttm group. The current study results indicated that Baohe pill decoction improved diarrhea symptoms by regulating the balance of mucosal microbiota. Specifically, Baohe pill decoction increased the abundance of probiotic Lactobacillus and decreased the abundance of the opportunistic Ralstonia pathogen.

Inflammation Controls Susceptibility of Immune-Experienced Mice to Sepsis.

Sepsis, an amplified immune response to systemic infection that leads to life-threatening organ dysfunction, affects >125,000 people/day worldwide with 20% mortality. Modest therapeutic progress for sepsis has been made, in part because of the lack of therapeutic translatability between mouse-based experimental models and humans. One potential reason for this difference stems from the extensive use of immunologically naive specific pathogen-free mice in preclinical research. To address this issue, we used sequential infections with well-defined BSL-2 pathogens to establish a novel immune-experienced mouse model (specific pathogen experienced [SPexp]) to determine the extent to which immunological experience and/or inflammation influences the host capacity to respond to subsequent infections, including sepsis. Consistent with their immunological experience, SPexp inbred or outbred mice had significant changes in the composition and activation status of multiple leukocyte populations known to influence the severity of cecal ligation and puncture-induced sepsis. Importantly, by varying the timing of sepsis induction, we found the level of basal inflammation controls sepsis-induced morbidity and mortality in SPexp mice. In addition, although a beneficial role of NK cells in sepsis was recently demonstrated in specific pathogen-free mice, NK cell depletion before cecal ligation and puncture induction in SPexp mice lead to diminished mortality, suggesting NK cells may have beneficial or detrimental roles in the response to septic insult dependent on host immune status. Thus, data highlight the importance of utilizing immune-experienced models for preclinical studies to interrogate the cellular/molecular mechanism(s) that could be therapeutically exploited during severe and dysregulated infection-induced inflammatory responses, such as sepsis.

Using Filter Media and Soiled Bedding in Disposable Individually Ventilated Cages as a Refinement to Specific Pathogen-free Mouse Health Monitoring Programs.

Molecular-based methods have shown potential for improving pathogen detection and reducing animal use. While increasing evidence supports rodent-free environmental health PCR pathogen detection, limited information is available regarding efficacy for disposable individually ventilated caging systems. In such systems, testing of plenum exhaust air dust is ineffective, and the use of collection media is optimal. We performed a series of studies to compare PCR infectious agent detection with dust collected on media placed in a mouse-free soiled bedding cage, the cage exhaust filter of an occupied sentinel cage, and direct sampling from colony and sentinel mice with traditional soiled bedding mouse sentinels. We hypothesized that after a 3-mo period, testing of filter media agitated in a soiled bedding cage would be equal to or more sensitive than more traditional methods. Agitated media detected Astrovirus-1, segmented filamentous bacteria and Helicobacter ganmani to a degree comparable to testing lid exhaust filter PCR from a sentinel mouse cage, but opportunists such as Staphylococcus aureus and Proteus mirabilis were not detected consistently, and H. hepaticus was not detected at all. Direct sampling of pooled fecal pellets and body swabs from sentinel mice and testing using PCR also failed to reliably detect opportunists and Helicobacter spp. While further work is needed to refine use of filter media in soiled bedding for detection of lower prevalence opportunists, this report provides evidence that a rodent-free method of reliably detecting murine agents in a disposable individually ventilated cage system with cage-level filtration outperforms direct sampling of soiled bedding sentinel mice.

A Microbiota-Dependent Subset of Skin Macrophages Protects Against Cutaneous Bacterial Infection.

Microbiota is essential to the development and functional maturation of the immune system. The effects of the gut microbiota on myeloid cells remote from the gut, especially the skin remain unclear. Transcriptomic analysis revealed that type I interferon (IFN) signaling was down-regulated in the skin of germ-free mice compared to that in specific pathogen-free mice. The decrease in type I IFN signaling was closely related to the presence of microbiota and macrophage-specific marker CD169. The absence of CD169+ macrophages resulted in increased bacterial burden and impaired immune responses against Staphylococcus aureus skin infection. CD169+ macrophages mediated the recruitment of γδ T cells as well as the activation of γδ T cells via interleukin (IL)-23. Our findings demonstrate the role of the microbiota in establishment of a specific myeloid cell subset expressing CD169 in the skin and provide evidence of a specific mechanism by which this subset protects against bacterial skin infection.

Microbial regulation of intestinal motility provides resistance against helminth infection

Soil-transmitted helminths cause widespread disease, infecting ~1.5 billion people living within poverty-stricken regions of tropical and subtropical countries. As adult worms inhabit the intestine alongside bacterial communities, we determined whether the bacterial microbiota impacted on host resistance against intestinal helminth infection. We infected germ-free, antibiotic-treated and specific pathogen-free mice, with the intestinal helminth Heligmosomoides polygyrus bakeri. Mice harboured increased parasite numbers in the absence of a bacterial microbiota, despite mounting a robust helminth-induced type 2 immune response. Alterations to parasite behaviour could already be observed at early time points following infection, including more proximal distribution of infective larvae along the intestinal tract and increased migration in a Baermann assay. Mice lacking a complex bacterial microbiota exhibited reduced levels of intestinal acetylcholine, a major excitatory intestinal neurotransmitter that promotes intestinal transit by activating muscarinic receptors. Both intestinal motility and host resistance against larval infection were restored by treatment with the muscarinic agonist bethanechol. These data provide evidence that a complex bacterial microbiota provides the host with resistance against intestinal helminths via its ability to regulate intestinal motility.

Maternal Administration Of Probiotic Lactobacilli Since Pre-Conception Programs Offspring Gut Microbiota Functions

ObjectivesProbiotics intake may be considered beneficial by prospective and pregnant mothers but their effects on offspring development are underexplored. The maternal gut microbiota is a determinant of early offspring gut microbial colonization patterns and pre-conception may be a critical period. Our aim was to assess the programming effects of a probiotic Lacticaseibacillus rhamnosus R0011 and Lactobacillus helveticus R0052 mixture, which is known to support intestinal homeostasis in infancy, on the temporal dynamics of offspring gut microbiota functions.MethodsFemale C57BL/6 specific pathogen free mice received sterile or probiotic-supplemented water starting one week before mating and until weaning (postnatal day, PND, 21). Offspring remained on their respective mother's treatment from PND 21 to 36. Offspring cecal microbiota was profiled at PND 14, 21, and 36 by 16S rRNA (V3-V4) gene amplicon sequencing and metabolic pathways were inferred with PICRUSt2 and the MetaCyc database. Data analysis and clustering were conducted with DESeq2-Wald test and hclust in R.Results355 MetaCyc pathways were predicted and collapsed into 48 functional classes; 10 of these accounted for more than 80% of the total pathway relative abundance, including various biosynthetic as well as carbohydrate and protein degradation pathways. With time, 113 and 55 pathways were altered (q-values < 0.01) in the control and probiotic groups, respectively, but only control samples clustered by age. At PND 14 and 21,195 and 39 pathways were differentially represented in the probiotic group vs. controls, but none at PND 36. Age-dependent pathways altered by probiotic supplementation belonged to biosynthesis (Amino Acid, Nucleoside and Nucleotide, Cofactor, Electron Carrier, and Vitamin) and degradation (Carbohydrate, Nucleoside and Nucleotide, Secondary Metabolite Degradation, C1 Compound Utilization and Assimilation) functional classes.ConclusionsMaternal probiotic supplementation accelerates the establishment of gut microbiota metabolic functions necessary to process host diet as well as for the synthesis of metabolites relevant for host health in the juvenile offspring.Funding SourcesNSERC; Lallemand Health Solutions; Lawson Family Chair in Microbiome Research.

Alleviation of oxidative stress in pancreatic tissue of hyperglycemic mice by Lactiplantibacillus plantarum SCS4.

This study aimed to investigate the effect of Lactiplantibacillus plantarum SCS4 in alleviating oxidative stress in the pancreatic tissue of hyperglycemic mice. A total of 90 six-week-old specific pathogen-free male Kunming mice were randomly divided into six groups [normal group (NG), blank control group (MG), phosphate-buffered saline (PBS) control group (CG), SCS4 first control group (DT1), SCS4 second control group (DT2), and SCS4 third control group (DT3)]. Except the NG group, in the other five groups, streptozotocin (STZ) was intraperitoneally injected for five consecutive days to establish a hyperglycemia model; the concentration of STZ was 50 mg/kg (bw). After successful modeling, DT1, DT2, and DT3 mice were administered 0.2ml of L. plantarum SCS4 bacterial solution (1010 colony forming unit/ml), the cellular content of L. plantarum SCS4, and the inactivated cellular content of L. plantarum SCS4, respectively. Furthermore, 0.2ml of PBS was given to mice in the CG group for control. The levels of insulin (INS), reactive oxygen species (ROS), malondialdehyde (MDA), and antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured by enzyme-linked immunosorbent assay, and the morphology of the pancreas was observed. The results showed that after 10 weeks of gavage treatment, the level of INS in the DT3 group significantly increased to 6.36 mIU/L compared with that in the MG group (p < .05). Meanwhile, the levels of ROS and MDA of DT3 returned to normal levels of 291.07 IU/ml and 4.29 mnol/L, respectively. The activities of various antioxidant enzymes increased. The levels of SOD, CAT, and GPx in DT3 were the closest to the levels in NG. In addition, the pathological sections showed that the degree of pancreatic tissue lesions was relatively more severe in the MG group than in the NG group. The degree of pancreatic tissue lesions was relatively less severe in the DT2 group than in the MG group, and no significant lesion was seen in the DT3 group. Our results indicated that the inactivated bacterial content of L. planetarium SCS4 was more effective in improving oxidative stress in the pancreas of hyperglycemic mice. PRACTICAL APPLICATIONS: L.plantarum SCS4 was separated from fermented sausage in Sichuan. This study indicated that inactivated bacterial content of L. planetarium SCS4 was more effective in improving oxidative stress in the pancreas of hyperglycemic mice. The results suggested that lactic acid bacteria from traditional foods with ability of improving oxidative damage, which can be applied in food nutrition and related fields to make people with good dietary habits and prevent the occurrence of chronic diseases such as type II diabetes effectively.

Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction.

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses’ whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

PD32-07 MICROBIAL EXPOSURE REDUCES SUSCEPTIBILITY TO UROPATHOGENIC E. COLI (UPEC)-INDUCED UROSEPSIS

You have accessJournal of UrologyCME1 May 2022PD32-07 MICROBIAL EXPOSURE REDUCES SUSCEPTIBILITY TO UROPATHOGENIC E. COLI (UPEC)-INDUCED UROSEPSIS Matthew Martin, Whitney Swanson, Tamara Kucaba, Vladimir Badovinac, Molly Ingersoll, and Thomas Griffith Matthew MartinMatthew Martin More articles by this author , Whitney SwansonWhitney Swanson More articles by this author , Tamara KucabaTamara Kucaba More articles by this author , Vladimir BadovinacVladimir Badovinac More articles by this author , Molly IngersollMolly Ingersoll More articles by this author , and Thomas GriffithThomas Griffith More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002583.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary tract infections (UTIs) are the most prevalent infectious disease of the bladder – affecting >130 million people worldwide annually. Uropathogenic E. coli (UPEC) causes most UTIs, starting with bladder colonization and then ascension to the kidneys. In severe cases of pyelonephritis, bacteria can enter the bloodstream, causing urosepsis. The immune system is involved in the development of these pathologies, but little is known about how the basal immune state influences the ability to respond to systemic UPEC infection (urosepsis). Most preclinical studies are conducted with specific pathogen-free (SPF) mice, which possess a largely naïve immune system like neonatal humans. Cohousing (CoH) SPF mice with microbially diverse pet store mice matures the immune system to resemble that of adult humans. Thus, the objective of these studies is to determine how prior microbial exposure affects the immune response to systemic UPEC infection modeling urosepsis. METHODS: For these studies, we infected SPF and CoH mice with UPEC strain UTI89 systemically via intravenous injection. Immune responses, bacterial clearance, and survival following infection was monitored. RESULTS: Interestingly, we found CoH were less susceptible to mortality following systemic infection and cleared UPEC at a faster rate than SPF mice. Following infection, greater numbers of neutrophils, macrophages, CD4 T cells, and antigen-experienced CD8 T cells, and higher amounts of inflammatory cytokines including IFNg were found in the circulation of CoH mice vs. SPF mice. Upon immune cell subset depletion, macrophage-depleted CoH mice showed enhanced mortality to infection similar to SPF mice, and macrophage-depleted CoH mice cleared infection at a slower rate than intact CoH mice, suggesting macrophages are responsible for enhanced protection from systemic UPEC infection in CoH mice. RNA sequencing of macrophages from SPF and CoH mice revealed that microbial exposure alters the macrophage transcriptional profile and suggested cohousing results in a program of innate immunity training. CONCLUSIONS: Our results have important implications in the study of urosepsis, as the data suggest a general history of microbial exposure dramatically alters innate immunity and reduces susceptibility to systemic UPEC infection. While these studies further our understanding of the immune response initiated after systemic UPEC infection, they also suggest mouse studies incorporating exposure to diverse infections may provide a more clinically relevant experimental urosepsis model. Source of Funding: American Urological Association Research Scholar Award (MDM) NIH R21 AI154527-01A1 (TSG) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e549 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Martin More articles by this author Whitney Swanson More articles by this author Tamara Kucaba More articles by this author Vladimir Badovinac More articles by this author Molly Ingersoll More articles by this author Thomas Griffith More articles by this author Expand All Advertisement PDF DownloadLoading ...

Kidney and Gut Microbiome Crosstalk in Response to High Dietary Potassium

In this study, we tested the hypothesis that the gut microbiome influences the homeostatic response to a dietary K+ load. Male Specific Pathogen Free (SPF) and Germ‐Free (GF) mice (n = 5 per group) received 5% potassium citrate or K+alimentary matched control diets for 7 days. At the time of sacrifice, blood and urine parameters were compared, and the expression of relevant transporters and signaling molecules was assessed in the kidney by western blotting. No significant differences in whole blood K+ were observed between SPF and GF mice on control or high K+ diets. However, differences in other parameters were observed. Compared to control diet treated SPF mice, K+ loaded SPF mice exhibited a significant decrease in hematocrit (37 vs 39; p ≤ 0.0152) and hemoglobin (12.58 vs 13.26; p ≤ 0.0150), whereas GF mice exhibited no differences in these parameters regardless of dietary maneuver. Blood urea nitrogen (BUN) levels were significantly decreased in SPF mice (23.4mg/dL vs 27; p ≤ 0.0180) on High K+compared to SPF controls, while this was not the case for GF mice. These findings suggest that K+ loaded GF mice were less volume expanded than K+ loaded SPF mice. Consistent with this, plasma aldosterone measurements were higher in K+ loaded GF mice compared to K+ loaded SPF mice (966pg/mL vs 614; p ≤ 0.0155). In whole kidney homogenates, dietary K+ loading markedly increased the cleaved (active) form of the epithelial sodium channel gamma subunit (γENaC) relative to control diet treated mice in both the SPF and GF groups. However, cleaved γENaC was one‐fold higher in the K+ loaded GF mice compared to K+ loaded SPF mice. K+ citrate loading significantly upregulated pendrin in both GF and SPF groups (p ≤ 5.7146E‐05 and p ≤ 5.6654E‐06), likely an effect of the alkaline content of the diet. Interestingly, we also observed a significant 91% increase in Aquaporin 2 (AQP2) levels in the GF high K+ group (p ≤ 0.0168) relative to GF mice treated with control diet, an effect that was not observed in SPF mice (p= 0.165). These findings indicate that male germ‐free mice maintain plasma [K+] in response to a dietary alkaline potassium load, but develop reduced volume status compared to K+ loaded SPF controls. To compensate, germ free mice exhibit plasma elevated aldosterone concentrations, and upregulate cleaved γENaC and AQP2 abundance in the kidney. This suggests that an intact gut microbiome helps to defend against excessive volume depletion during the homeostatic response to alkaline K+ loading.

Expansion and maintenance of long lived effector CD8 T cells is modulated by inflammation

Abstract Long-lived effector T cells (LLEC) are a circulating population of KLRG1 and CX3CR1 expressing memory CD8 T cells. LLEC were first described by the Hamilton Lab and are highly effective at clearing systemic infections compared to other memory T cells. However, LLEC express lower levels of IL-7 and IL-15 receptor chains and are poorly proliferative in specific pathogen free (SPF) mice as compared to central and effector memory T cells, which leads to a slow attrition of their numbers. The factors supporting LLEC expansion and persistence are unknown, and the goal of this study is to understand what internal and external factors contribute to LLEC expansion and maintenance. Data from models of chronic inflammation suggest pro-inflammatory factors contribute to the expansion and/or survival of LLEC. Specifically, we have found that LLEC populations are increased in young mice that have been exposed to a diverse mixture of microbes through co-housing with pet-shop mice. This co-housing model chronically increases systemic inflammation, and LLEC specific for model antigens not only expand more, but also persist longer than in SPF mice. Similarly, we also find increased LLEC in aged mice, another model associated with chronic inflammation. In preliminary work, we find that LLEC division is increased during cohousing, and that IL-18R expression is elevated, further suggesting that inflammatory cytokines not typically associated with T cell persistence may support LLEC proliferation. Ongoing studies will determine which specific inflammatory markers support LLEC expansion and maintenance, whether these factors are detrimental to other memory subsets, and if this process can be altered to improve T cell responses to infection and vaccination. Supported by grants from NIH (R01 AI143828).