Congenic Research Articles

The H2-A Class II molecule α/β-chain cis-mismatch severely affects cell surface expression, selection of conventional CD4+ T cells and protection against TB infection.

To dissect the role of the part of the H2 complex comprised of the MHC-II genes in the control of tuberculosis (TB) infection, we previously established a panel of recombinant congenic mouse strains bearing different segments of the H2 j haplotype on the B6 (H2 b) genetic background. Fine genetic mapping, gene sequencing and assessment of TB phenotypes resulted in identification of the H2-Ab gene as a major factor of TB control. We further narrowed the MHC-II H2 j interval by spotting a new recombination event, sequencing newly established DNA configuration and establishing a mouse strain B6.I-103 in which j/b recombination occurred within the coding sequence of the H2-Ab gene. Unexpectedly, a novel H2-Aα b/AβjE0 haplotype provided exclusively high susceptibility to TB challenge. Immunologic analysis revealed an altered CD4+ T-cell selection and maintenance in B6.I-103 mice, as well as seriously impaired expression of the H2-Aαb/Aβj molecule on the surface of antigen presenting cells. Unlike previously reported cases of Class II malfunctioning, the defective phenotype arose not from strong structural mutations, but from regular recombination events within the MHC-II recombination hot spot region. Our findings provide evidence that Class II α/β-chain cis-allelic mismatches created by regular genetic recombination may severely affect immune system functioning. This issue is discussed in the context of the MHC evolution.

#4127 MODULATION OF BLOOD PRESSURE THROUGH MICROBIOME EXCHANGE BETWEEN MILAN NORMOTENSIVE AND HYPERTENSIVE STRAINS OF RATS

Abstract Background and Aims Hypertension is one of the major health problems contributing to the development of cardiovascular diseases. In contrast to essential hypertension, which often displays complex pathogenesis due to multifactorial aetiology, several monogenic forms of hypertension are caused by mutations in genes associated with renal salt handling. Thus, studies on monogenic forms of hypertension could hold potential for novel therapeutic approaches for essential hypertension. Recently, increasing numbers of studies have demonstrated the alteration of the gut microbiome in pathological states including hypertension. These observations suggest the involvement of the intestinal microbiome in the development and/or maintenance of hypertension. A reciprocal congenic strain of Milan hypertensive rat (NA rat), harbouring a missense mutation in the gene encoding the alpha subunit of the membrane-skeletal protein adducin (Add1), develops hypertension starting from 3 months after birth. Preliminary studies on NA rats and its littermate Milan normotensive (MN) rats showed diverse expression patterns of renal sodium transporters/ion channels in two strains of rats, indicating a putative role of altered renal salt handling on the enhanced blood pressure in NA rats (unpublished data). Based on this background, the aim of this study was to investigate a possible connection between the gut microbiome and blood pressure phenotypes in these two strains. Furthermore, we evaluated whether the exchange of faeces between the two strains could transfer the phenotypes through the gut microbiome. Finally, mechanisms underlying the altered phenotype after microbiome exchange were investigated. Method Hypertensive NA rats and normotensive MN rats were subjected to ‘homogenization (HOM)’ of the microbiome after birth, which consisted of the exchange of bedding containing faeces from either strain, followed by co-housing of MN and NA rats in the same cage (MN-HOM and NA-HOM groups). For the baseline (BSL) condition, rats were homogenized within the same strain (MN-BSL and NA-BSL). At the age of 5 months, blood pressure measurement was performed using a non-invasive tail-cuff BP-2000 Blood Pressure Analysis system. Faeces from each rat were collected for microbiota metataxonomic analysis using next generation sequencing of 16S ribosome encoding DNA. At the end of the treatment, rats were sacrificed for the collections of blood and organs to assess the expressions of genes involved in renal salt reabsorption. Results Systolic blood pressure (SBP) in MN-BSL was averaged at 143.6 mmHg, while NA-BSL showed average SBP 163.3 mmHg, confirming the hypertensive phenotype in NA rats at baseline (P<0.001). Interestingly, MN-HOM showed a significantly enhanced SBP compared to MN-BSL, reaching 153.5 mmHg (P = 0.005). On the other hand, the average SBP of NA-HOM was163.7 mmHg, indicating that homogenization did not significantly impact blood pressure in NA rats. Although the MN-HOM group showed significantly increased SBP compared with MN-BSL group, the average SBP of MN-HOM was still significantly lower than both the NA-BSL and NA-HOM groups. Different gut microbiota compositions were observed in the two strains of rats at baseline, and HOM altered both. Conclusion We have demonstrated that the hypertensive phenotype in NA rats was transferred to normotensive MN rats through the exchange of gut microbiota, indicating that the intestinal microbiota and/or metabolites derived therefrom could eventually modulate blood pressure. Further studies including immunoblotting as well as uremic toxin analyses are required to unveil the molecular mechanisms by which the gut microbiome modulates blood pressure.

Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions

Abstract Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).

The Role of C1473G Polymorphism in Mouse Triptophan Hydroxylase 2 Gene in the Acute Effects of Ethanol on the c-fos Gene Expression and Metabolism of Biogenic Amines in the Brain

Tryptophan hydroxylase 2 is a key enzyme in the synthesis of the neurotransmitter serotonin, which plays an important role in the regulation of behavior and various physiological functions. We studied the effect of acute ethanol administration on the expression of the early response c-fos gene and metabolism of serotonin and catecholamines in the brain structures of B6-1473C and B6-1473G congenic mouse strains differing in the single-nucleotide substitution C1473G in the Tph2 gene and activity of the encoded enzyme. Acute alcoholization led to a significant upregulation of the c-fos gene expression in the frontal cortex and striatum of B6-1473G mice and in the hippocampus of B6-1473C mice and caused a decrease in the index of serotonin metabolism in the nucleus accumbens in B6-1473C mice and in the hippocampus and striatum of B6-1473G mice, as well as to the decrease in the norepinephrine level in the hypothalamus of B6-1473C mice. Therefore, the C1473G polymorphism in the Tph2 gene has a significant effect of acute ethanol administration on the c-fos expression pattern and metabolism of biogenic amines in the mouse brain.

Dynamic NIR Fluorescence Imaging and Machine Learning Framework for Stratifying High vs. Low Notch-Dll4 Expressing Host Microenvironment in Triple-Negative Breast Cancer.

Delta like canonical notch ligand 4 (Dll4) expression levels in tumors are known to affect the efficacy of cancer therapies. This study aimed to develop a model to predict Dll4 expression levels in tumors using dynamic enhanced near-infrared (NIR) imaging with indocyanine green (ICG). Two rat-based consomic xenograft (CXM) strains of breast cancer with different Dll4 expression levels and eight congenic xenograft strains were studied. Principal component analysis (PCA) was used to visualize and segment tumors, and modified PCA techniques identified and analyzed tumor and normal regions of interest (ROIs). The average NIR intensity for each ROI was calculated from pixel brightness at each time interval, yielding easily interpretable features including the slope of initial ICG uptake, time to peak perfusion, and rate of ICG intensity change after reaching half-maximum intensity. Machine learning algorithms were applied to select discriminative features for classification, and model performance was evaluated with a confusion matrix, receiver operating characteristic curve, and area under the curve. The selected machine learning methods accurately identified host Dll4 expression alterations with sensitivity and specificity above 90%. This may enable stratification of patients for Dll4 targeted therapies. NIR imaging with ICG can noninvasively assess Dll4 expression levels in tumors and aid in effective decision making for cancer therapy.

E96V Mutation in the Kdelr3 Gene Is Associated with Type 2 Diabetes Susceptibility in Obese NZO Mice.

Type 2 diabetes (T2D) represents a multifactorial metabolic disease with a strong genetic predisposition. Despite elaborate efforts in identifying the genetic variants determining individual susceptibility towards T2D, the majority of genetic factors driving disease development remain poorly understood. With the aim to identify novel T2D risk genes we previously generated an N2 outcross population using the two inbred mouse strains New Zealand obese (NZO) and C3HeB/FeJ (C3H). A linkage study performed in this population led to the identification of the novel T2D-associated quantitative trait locus (QTL) Nbg15 (NZO blood glucose on chromosome 15, Logarithm of odds (LOD) 6.6). In this study we used a combined approach of positional cloning, gene expression analyses and in silico predictions of DNA polymorphism on gene/protein function to dissect the genetic variants linking Nbg15 to the development of T2D. Moreover, we have generated congenic strains that associated the distal sublocus of Nbg15 to mechanisms altering pancreatic beta cell function. In this sublocus, Cbx6, Fam135b and Kdelr3 were nominated as potential causative genes associated with the Nbg15 driven effects. Moreover, a putative mutation in the Kdelr3 gene from NZO was identified, negatively influencing adaptive responses associated with pancreatic beta cell death and induction of endoplasmic reticulum stress. Importantly, knockdown of Kdelr3 in cultured Min6 beta cells altered insulin granules maturation and pro-insulin levels, pointing towards a crucial role of this gene in islets function and T2D susceptibility.

Elimination of 4T1 Mammary Tumor Cells by BALB/cBy UBC-GFP Transgenics following Stable Inheritance of the H-2b MHC Allele.

The human ubiquitin C promoter (UBC)-driven GFP-transgenic mouse (UBC-GFP) transgene integration site was mapped recently to chromosome 17, linked closely to the MHC locus. In this study, we demonstrate a functional consequence of this insertion site in the backcrossed UBC-GFP BALB/c congenic strain [CByJ.B6-Tg(UBC-GFP) 30Scha/J]: rejection of transplanted "syngeneic" 4T1 mammary tumor cells. Rejection of BALB/c-derived 4T1 cells is in all likelihood a consequence of MHC mismatch due to stable inheritance of C57BL/6-derived H-2b (rather than prototypical H-2d) by the BALB/c UBC-GFP strain. These data are a valuable resource to researchers who have previously employed the UBC-GFP congenic strain for attempted syngeneic MHC-matched and allogenic MHC-mismatched studies, as their data likely require reinterpretation. Further, this study reemphasizes the impact of mapping transgene integration sites of commonly used mouse strains as a way of increasing scientific rigor and reproducibility.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

Rat Mammary carcinoma susceptibility 3 (Mcs3) pleiotropy, socioenvironmental interaction, and comparative genomics with orthologous human 15q25.1-25.2.

Genome-wide association studies of breast cancer susceptibility have revealed risk-associated genetic variants and nominated candidate genes; however, the identification of causal variants and genes is often undetermined by genome-wide association studies. Comparative genomics, utilizing Rattus norvegicus strains differing in susceptibility to mammary tumor development, is a complimentary approach to identify breast cancer susceptibility genes. Mammary carcinoma susceptibility 3 (Mcs3) is a Copenhagen (COP/NHsd) allele that confers resistance to mammary carcinomas when introgressed into a mammary carcinoma susceptible Wistar Furth (WF/NHsd) genome. Here, Mcs3 was positionally mapped to a 7.2-Mb region of RNO1 spanning rs8149408 to rs107402736 (chr1:143700228-150929594, build 6.0/rn6) using WF.COP congenic strains and 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis. Male and female WF.COP-Mcs3 rats had significantly lower body mass compared to the Wistar Furth strain. The effect on female body mass was observed only when females were raised in the absence of males indicating a socioenvironmental interaction. Furthermore, female WF.COP-Mcs3 rats, raised in the absence of males, did not develop enhanced lobuloalveolar morphologies compared to those observed in the Wistar Furth strain. Human 15q25.1-25.2 was determined to be orthologous to rat Mcs3 (chr15:80005820-82285404 and chr15:83134545-84130720, build GRCh38/hg38). A public database search of 15q25.1-25.2 revealed genome-wide significant and nominally significant associations for body mass traits and breast cancer risk. These results support the existence of a breast cancer risk-associated allele at human 15q25.1-25.2 and warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 to discover novel causal genes and variants.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time pointspost-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6×BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Two Loci Contribute to Age-Related Hearing Loss Resistance in the Japanese Wild-Derived Inbred MSM/Ms Mice

An MSM/Ms strain was established using Japanese wild mice, which exhibit resistance to several phenotypes associated with aging, such as obesity, inflammation, and tumorigenesis, compared to common inbred mouse strains. MSM/Ms strain is resistant to age-related hearing loss, and their auditory abilities are sustained for long durations. The age-related hearing loss 3 (ahl3) locus contributes to age-related hearing in MSM/Ms strain. We generated ahl3 congenic strains by transferring a genomic region on chromosome 17 from MSM/Ms mice into C57BL/6J mice. Although C57BL/6J mice develop age-related hearing loss because of the ahl allele of the cadherin 23 gene, the development of middle- to high-frequency hearing loss was significantly delayed in an ahl3 congenic strain. Moreover, the novel age-related hearing loss 10 (ahl10) locus associated with age-related hearing resistance in MSM/Ms strain was mapped to chromosome 12. Although the resistance effects in ahl10 congenic strain were slightly weaker than those in ahl3 congenic strain, slow progression of age-related hearing loss was confirmed in ahl10 congenic strain despite harboring the ahl allele of cadherin 23. These results suggest that causative genes and polymorphisms of the ahl3 and ahl10 loci are important targets for the prevention and treatment of age-related hearing loss.

Abstract P319: Genetic Interactions Between Novel Genes Arhgef11 And Cgnl1 Are Involved In Hypertensive Chronic Kidney Disease

Patients with hypertension are at higher risk for developing chronic kidney disease (CKD) and ultimately kidney failure. Previous genetic studies involving the Dahl salt-sensitive (SS) rat, a well-established model of hypertensive CKD, identified multiple regions linked to kidney injury. Extensive work on Chr.2 identified and characterized the role of Arhgef11 in the onset and progression of CKD using SS- Arhgef11 -/- rats. The identification of Arhgef11 and mechanism (regulation of actin cytoskeleton components) has provided an opportunity to expedite gene identification on other chromosomes based on known genetic interactions between loci, such as with Chr. 8. The effect of the Chr. 8 locus on kidney injury was previously validated using a congenic strain [S.SHR(8)]. Bioinformatics analysis of genes residing within the confidence interval, and those mechanistically linked with Arhgef11 , identified cingulin-like 1 ( Cgnl1 ) as a candidate. Cgnl1 plays a role in the regulation of cell-cell contacts via adherens and tight junctions through several interacting proteins [e.g. ZO-1 and PLEKHA7 (linked with hypertension by GWAS and an SS- Plekha7 mutant model)]. Recent studies using a novel model (SS- Cgnl1 -/- ) demonstrates that knockout of Cgnl1 leads to loss of salt-sensitive blood pressure and reduced renal injury compared to wildtype SS, supporting the congenic studies. Using multi-omics approaches incorporating bulk RNAseq, single nuclei RNAseq, spatial transcriptomics, small RNAseq, and mass spectrometry discovery proteomics, a putative mechanism connecting proximal tubule injury to alterations in sodium solute transporters and hypertension was established. The direct physiological interaction between Arhgef11 and Cgnl1 on hypertension and CKD is currently underway using a double knockout model (SS- Arhgef11 -/- Cgnl1 -/- ). In addition, to better elucidate the impact of loss of Arhgef11 , Cgnl1 , or both genes together, gene-edited cell lines using CRISPR technology has been generated and are also being studied. It is anticipated that completion of this work will lead to a better understanding of the role of genetics in hypertensive CKD, the influence of factors that complicate kidney disease, and potential new therapeutic targets.

Nutrigenetic Interaction of Spontaneously Hypertensive Rat Chromosome 20 Segment and High-Sucrose Diet Sensitizes to Metabolic Syndrome.

Several corresponding regions of human and mammalian genomes have been shown to affect sensitivity to the manifestation of metabolic syndrome via nutrigenetic interactions. In this study, we assessed the effect of sucrose administration in a newly established congenic strain BN.SHR20, in which a limited segment of rat chromosome 20 from a metabolic syndrome model, spontaneously hypertensive rat (SHR), was introgressed into Brown Norway (BN) genomic background. We mapped the extent of the differential segment and compared the genomic sequences of BN vs. SHR within the segment in silico. The differential segment of SHR origin in BN.SHR20 spans about 9 Mb of the telomeric portion of the short arm of chromosome 20. We identified non-synonymous mutations e.g., in ApoM, Notch4, Slc39a7, Smim29 genes and other variations in or near genes associated with metabolic syndrome in human genome-wide association studies. Male rats of BN and BN.SHR20 strains were fed a standard diet for 18 weeks (control groups) or 16 weeks of standard diet followed by 14 days of high-sucrose diet (HSD). We assessed the morphometric and metabolic profiles of all groups. Adiposity significantly increased only in BN.SHR20 after HSD. Fasting glycemia and the glucose levels during the oral glucose tolerance test were higher in BN.SHR20 than in BN groups, while insulin levels were comparable. The fasting levels of triacylglycerols were the highest in sucrose-fed BN.SHR20, both compared to the sucrose-fed BN and the control BN.SHR20. The non-esterified fatty acids and total cholesterol concentrations were higher in BN.SHR20 compared to their respective BN groups, and the HSD elicited an increase in non-esterified fatty acids only in BN.SHR20. In a new genetically defined model, we have isolated a limited genomic region involved in nutrigenetic sensitization to sucrose-induced metabolic disturbances.

The influence of chromosome 4 on high ethanol consumption and blood pressure

The Spontaneously Hypertensive Rats (SHR) strain was developed through selective breeding for high systolic blood pressure. In our laboratory, we established a congenic rat strain named SHR.Lewis-Anxrr16 (SLA16). The SLA16 rat strain is genetically identical to the SHR except for the inserted Anxrr16 region in chromosome 4. Our objective was to evaluate the influence of this genomic region on ethanol consumption and blood pressure. First, we exposed SHR and SLA16 male and female rats to ethanol consumption. Results showed that, regardless of strain, females consumed more ethanol than males during forced (10% v/v) and spontaneous ethanol consumption (SEC; 2.5-20% v/v). Then, females from both strains were used to evaluate sensitivity to ethanol. No strain differences in the loss of righting reflex were observed after ethanol treatment (3g/kg, 20% w/v, intraperitoneal [i.p.]). But, in the triple test, female SHR rats presented lower sensitivity to the ethanol (1.2g/kg, 14% w/v, i.p.). Surprisingly, female SHR rats also presented higher blood pressure after SEC (10% v/v). Finally, losartan treatment was effective in decreasing the blood pressure of female rats of both strains, but had specific effects on SHR ethanol consumption. Our data suggest that SLA16 female rats consume less ethanol (10%), are more sensitive to its effects, and present lower blood pressure than SHR female rats. We demonstrated that the Anxrr16 locus in chromosome 4 is a genetic candidate to explain high ethanol consumption and blood pressure, at least in females.

Fine mapping and candidate gene analysis of a dravet syndrome modifier locus on mouse chromosome 11

Pathogenic variants in SCN1A result in a spectrum of phenotypes ranging from mild febrile seizures to Dravet syndrome, a severe infant-onset epileptic encephalopathy. Individuals with Dravet syndrome have developmental delays, elevated risk for sudden unexpected death in epilepsy (SUDEP), and have multiple seizure types that are often refractory to treatment. Although most Dravet syndrome variants arise de novo, there are cases where an SCN1A variant was inherited from mildly affected parents, as well as some individuals with de novo loss-of-function or truncation mutations that presented with milder phenotypes. This suggests that disease severity is influenced by other factors that modify expressivity of the primary mutation, which likely includes genetic modifiers. Consistent with this, the Scn1a+/− mouse model of Dravet syndrome exhibits strain-dependent variable phenotype severity. Scn1a+/− mice on the 129S6/SvEvTac (129) strain have no overt phenotype and a normal lifespan, while [C57BL/6Jx129]F1.Scn1a+/− mice have severe epilepsy with high rates of premature death. Low resolution genetic mapping identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival of Scn1a+/− mice. To confirm the Dsm5 locus and refine its position, we generated interval-specific congenic strains carrying 129-derived chromosome 11 alleles on the C57BL/6J strain and localized Dsm5 to a 5.9 Mb minimal region. We then performed candidate gene analysis in the modifier region. Consideration of brain-expressed genes with expression or coding sequence differences between strains along with gene function suggested numerous strong candidates, including several protein coding genes and two miRNAs that may regulate Scn1a transcript.

Beyond the gastrointestinal tract: oral and sex-specific skin microbiota are associated with hypertension in rats with genetic disparities.

Current knowledge of the link between microbiota and hypertension is limited to the gut. Besides the gut, oral cavity and skin are other locations where sodium chloride (NaCl) is in direct contact with microbiota. Although oral nitrate-reducing bacteria generate nitric oxide, which leads to vasodilation and lowering of blood pressure (BP), the skin excretes sodium via sweat glands and is an important site for sodium and BP homeostasis. However, knowledge on the contributions of oral and skin microbiota to BP regulation, is limited. Therefore, the current study was conducted to compare the tripartite relationship between site, sex, and genetic effects on the composition of oral, skin, and gut microbiota impacting hypertension. Microbiota were profiled from the oral cavity, skin, and feces of both male and female hypertensive Dahl salt-sensitive (S) and congenic rats with genomic substitutions on rat chromosomes (RNO) 1, 5, 9, and 10, demonstrating disparate BP effects. Sex-specific differences in β-diversity were observed only in skin microbiota. The most abundant taxa of the oral and skin microbiota were Actinobacteria and Cyanobacteria, respectively. Oral Actinobacteria were inversely associated with BP. Although the abundance of oral Actinobacteria was upregulated by the BP locus on RNO10 in both sexes, depletion of skin Cyanobacteria decreased the protection from hypertension in the RNO5 female, but not male, congenic strain. In conclusion, to our knowledge this is the first study to identify specific microbiota in sites other than gut as contributors to BP regulation. Notably, both oral Actinobacteria and skin Cyanobacteria were beneficial for lowering BP.

The Role of HnRNPA1 in T Cell-Mediated Gut Tolerance

Abstract Our previous research showed that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength, and occurs under conditions of induced T regulatory (Treg) cell differentiation. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is phosphorylated by Akt at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1), in which hnRNPA1 will no longer be Akt-phosphhorylated. Our initial characterization of mA1 mouse model failed to reveal major abnormalities in immune cells populations at steady state except for changes in the Treg proportion in the mesenteric lymph nodes. To investigate the effect of the mA1 on the development of oral tolerance we used an adoptive transfer model in which OT-II mA1 or OT-II WT naïve T cells were transferred into congenic strains and then the mice were given OVA food. Results indicate that mA1 OT-II T cells have a proliferation defect in vivo and we also observed a significant reduction in the induction of OVA-specific Treg in the mA1 T cells. Similar results were obtained when adoptively transferred OT-II mA1 T cells were challenged with limiting doses of specific peptide, given iv. RNASeq analysis of the transferred mA1 and WT T cells is being performed. These results suggest that Akt phosphorylation of hnRNPA1 is necessary for T cell response to low dose antigen and Treg induction in vivo. Our studies identify a new mechanism by which RBP influence T cell proliferation and Treg differentiation which has implications for tolerance to food antigens in the gut. Supported by NIH (F31-AI152320-02) NIH (R01-AI125513-03)

Beyond the Gastrointestinal Tract: Sex‐Specific Skin Microbiota Are Associated With Hypertension in Rats With Genetic Disparities

Current knowledge of microbiota in hypertension is restricted to the gut and mostly to males. Other than the kidney, the skin is the largest organ playing a role in sodium homeostasis and the mouth is the first site exposed to dietary salt, whereby, we hypothesized that microbiota in these sites associate with salt‐sensitive blood pressure (BP). To test this hypothesis, we compared microbiota from the skin, mouth and feces of both male and female young Dahl salt‐sensitive (S) rats and 4 genetically disparate congenic strains (RNO1, RNO5, RNO9 and RNO10). Skin microbiota was from 4‐6 days old rats before they developed their coats. Oral and fecal samples were collected at 4 weeks old. Microbiota were profiled by 16S rRNA sequencing. Compared to the gut, which is an anerobic site, microbiota from the aerobic skin and oral sites were consistently different in composition across all the strains. Unlike in the fecal samples where Firmicutes and Bacteroidetes were enriched, Cyanobacteria and Actinobacteria were enriched in the skin and oral samples, respectively. Female S rats had greater abundance of skin Cyanobacteria than male S rats. Strain effects were not observed in any of the strains or sites except for RNO5, where a strain × sex × site interactive effect was specifically noted for skin Cyanobacteria. Cyanobacteria contain sodium transporters and are salt‐tolerant microbiota, whereby they could potentially regulate sodium excretion and contribute to BP regulation. In summary, our study is the first to discover microbiota beyond the gut as being associated with BP.

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

A 3-Mbp fragment on rat chromosome 1 affects susceptibility both to stroke and kidney injury under salt loading in the stroke-prone spontaneously hypertensive rat: a genetic approach using multiple congenic strains.

We have previously reported that a major quantitative trait locus (QTL) responsible for susceptibility to salt-induced stroke in the stroke-prone spontaneously hypertensive rat (SHRSP) is located in a 3-Mbp region on chromosome 1 covered by SHRSP.SHR-(D1Rat23-D1Rat213)/Izm (termed Pr1.31), a congenic strain with segments from SHRSP/Izm introduced into the stroke-resistant SHR/Izm. Here, we attempted to narrow down the candidate region on chromosome 1 further through analyses of subcongenic strains constructed for the target region. Simultaneously, salt-induced kidney injury was evaluated through the measurement of urinary albumin and the gene expression of renal tubular injury markers (Kim-1 and Clu) to explore a possible mechanism leading to the onset of stroke. All subcongenic strains examined in this study showed lower susceptibility to salt-induced stroke than SHRSP. Interestingly, Pr1.31 had the lowest stroke susceptibility when compared with newly constructed subcongenic strains harboring fragments of the congenic sequence in Pr1.31. Although Kim-1 and Clu expression after 1 week of salt loading in Pr1.31 did not differ significantly from those in SHRSP, the urinary albumin level of Pr1.31 was significantly lower than those of the other subcongenic strains and that of SHRSP. The present results indicated that, although the congenic fragment in Pr1.31 harbored the gene(s) related to salt-induced organ damages, further genetic dissection of the candidate region was difficult due to multiple QTLs suggested in this region. Further analysis using Pr1.31 will unveil genetic and pathophysiological mechanisms underlying salt-induced end organ damages in SHRSP.