Modification of an aggressive model of Alport Syndrome reveals early differences in disease pathogenesis due to genetic background

The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modifed genetically. We have identifed a point mutation in Col4a4 in mice where disease is modifed by strain background, providing further evidence of the genetic modifcation of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identifed early diferences in disease progression, including expression of podocyte-specifc genes and podocyte morphology. In C57BL/6J mice podocyte efacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We fnd that there is evidence of diferential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an infammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function

Melody, an ENU mutation in Caspase 3, alters the catalytic cysteine residue and causes sensorineural hearing loss in mice

Progeny from the Harwell N-ethyl-N-nitrosourea (ENU) recessive mutagenesis screen were assessed for auditory defects. A pedigree was identified with multiple progeny lacking response to a clickbox test. Auditory brainstem response (ABR) analysis showed that homozygous mutant mice were profoundly deaf and the line was named melody. We subsequently mapped this mutation to a 6-Mb region on chromosome 8 and identified a point mutation in melody that results in a C163S substitution in the catalytic site of Caspase 3, a cysteine protease involved in apoptosis. Melody fails to complement a null Caspase-3 mutant. Scanning electron microscopy (SEM) has revealed disorganised sensory hair cells and hair cell loss. Histological analysis of melody has shown degeneration of spiral ganglion cells in homozygote mice, with a gradient of severity from apical to basal turns. Melody heterozygotes also show evidence of loss of spiral ganglion neurons, suggesting that the C163S mutation may show dominant negative effects by binding and sequestering proteins at the active site. The melody line provides a new model for studying the role of Caspase 3 in deafness and a number of other pathways and systems

T Regulatory Cells Control Susceptibility to Invasive Pneumococcal Pneumonia in Mice

Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-β between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-β protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3+Helios+ T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-β impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-β signalling is a potential target for immunotherapy or drug design

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss

Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep

AbstractThe circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep

The genetics of susceptibility to pneumococcal infection

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Spir2; a novel QTL on chromosome 4 contributes to susceptibility to pneumococcal infection in mice

BACKGROUND: Streptococcus pneumoniae causes over one million deaths worldwide annually, despite recent developments in vaccine and antibiotic therapy. Host susceptibility to pneumococcal infection and disease is controlled by a combination of genetic and environmental influences, but current knowledge remains limited. RESULTS: In order to identify novel host genetic variants as predictive risk factors or as potential targets for prophylaxis, we have looked for quantitative trait loci in a mouse model of invasive pneumococcal disease. We describe a novel locus, called Streptococcus pneumoniae infection resistance 2 (Spir2) on Chr4, which influences time to morbidity and the development of bacteraemia post-infection. CONCLUSIONS: The two quantitative trait loci we have identified (Spir1 and Spir2) are linked significantly to both bacteraemia and survival time. This may mean that the principle cause of death, in our model of pneumonia, is bacteraemia and the downstream inflammatory effects it precipitates in the host

IL-10-producing natural T regulatory cells are rapidly recruited to the lung in <i>S. pneumoniae</i>-infected BALB/c mice.

<p>All results are from intranasal-infection of mice with wild-type <i>S. pneumoniae</i> D39. (A) Foxp3 immunostaining of lung sections taken from BALB/c and CBA/Ca mice during pneumococcal infection. Inset shows example staining from 12 hour p.i. BALB/c mouse. Foxp3⁺ cells stain brown. (B) Number of Foxp3⁺ T regulatory cells, (C) Number of Foxp3⁺Helios⁺ cells, (D) number of Foxp3⁺IL-10⁺ cells and (E) number of Foxp3⁺CTLA-4⁺ cells per mg lung in <i>S. pneumoniae</i>-infected BALB/c and CBA/Ca mice, as identified by flow cytometry. Data in (D) are from 24 hrs p.i., PBS group contains both BALB/c and CBA/Ca mice. White bars=BALB/c, black bars=CBA/Ca. *'s indicate significant difference, where *=p<0.05, **=p<0.01, and ***=p<0.005. For all graphs data represent mean +/− SEM. All results are representative of between 2–4 independent experiments with >4 mice per group.</p

CBA/Ca mice display uncontrolled lung inflammation and associated apoptosis following <i>S. pneumoniae</i> infection.

<p>All results are from intranasal-infection of mice with wild-type <i>S. pneumoniae</i> D39. (A) Number of IFNγ⁺NKp46⁺CD4⁻ cells per mg lung in PBS-treated or <i>S. pneumoniae</i>-infected BALB/c and CBA/Ca mice at 24 hours p.i. (B) Top left shows gating of NK cells in lung homogenate. Remaining panels show example IFNγ-staining from PBS-treated CBA/Ca or <i>S. pneumoniae</i>-infected BALB/c and CBA/Ca mice at 24 hours p.i. (C) Proportion (%) of apoptotic cells in areas of inflammation within lungs of <i>S. pneumoniae</i>-infected BALB/c and CBA/Ca mice. Inset shows apoptotic cell (arrow) within area of inflammation as defined by presence of inflammatory infiltrate. White bars=BALB/c, black bars=CBA/Ca. *'s indicate significant difference, where *=p<0.05 and ***=p<0.005. For all graphs data represent mean +/− SEM. Results in (A and B) are representative of 3 independent experiments with >4 mice per group. Results in (C) are from a single experiment with 5 mice per group. PBS-treated groups contained both BALB/c and CBA/Ca mice.</p

Many <i>Tgfβ1</i> interaction partners show opposing expression profiles in BALB/c and CBA/Ca mice at 6 hours p.i.

<p>Ingenuity pathway analysis of microarray data from 6 hours p.i. Red=Gene significantly upregulated as compared to t=0 levels. Green=Down-regulated. BALB/c levels are compared to BALB/c t=0 expression and CBA/Ca levels are compared to CBA/Ca t=0 expression. Orange outline=Gene found within <i>Spir1</i> susceptibility-determining locus. Solid lines indicate direct interactions, dotted lines indicate indirect interactions.</p