Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Non-Typeable Haemophilus influenzae Infection of the Junbo Mouse.

Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non-typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host-microbial interactions relevant to the onset and progression of infection and to study treatment of middle ear disease. We have established a robust model of NTHi middle ear infection in the Junbo mouse. Intranasal inoculation with NTHi produces high rates of bulla infection and high bacterial titers in bulla fluids; bacteria can also spread down the respiratory tract to the mouse lung. An innate immune response is detected in the bulla of Junbo mice following NTHi infection, and bacteria are maintained in some ears at least up to day 56 post-inoculation. The Junbo/NTHi infection model facilitates studies on bacterial pathogenesis and antimicrobial intervention regimens and vaccines for better treatment and prevention of NTHi middle ear infection. © 2017 by John Wiley & Sons, Inc.

Myringotomy in the Junbo mouse model of chronic otitis media alleviates inflammation and cellular hypoxia.

OBJECTIVES/HYPOTHESIS: Ventilation of the chronically inflamed middle ear is a key outcome in functional middle ear surgery. Grommets eliminate middle ear effusion, but there is also evidence that they downregulate inflammation. The reason for this is not understood, but there is little to suggest alteration in eustachian tube ventilatory capacity. Previous work has shown that the Junbo mouse model of chronic otitis media has hypoxic middle ear mucosa and bulla fluid leucocytes. Here we explore whether surgical ventilation may alleviate chronic otitis media through downregulation of hypoxia. STUDY DESIGN: Surgical intervention on a mouse model of disease. METHODS: We established patency of myringotomy incision as 5 days in wild-type mice. We performed unilateral myringotomy on three cohorts of mice: 10 wild-type controls, 12 Junbo mice, and 15 Junbo mice with additional removal of middle ear effusion. A small cohort of these mice were labeled in vivo by intraperitoneal injection of pimonidazole to identify tissue hypoxia. Tissues were assessed for mucoperiosteal thickening and pimonidazole labeling, comparing operated to nonoperated ears. RESULTS: Ventilation of the inflamed Junbo middle ear revealed significant reduction in inflammatory thickening associated with loss of pimonidazole labeling, suggesting resolution of cellular hypoxia. CONCLUSIONS: Surgical ventilation may achieve therapeutic effect through alleviation of cellular hypoxia in the chronically inflamed middle ear. Targeted molecular therapy of hypoxia signaling may offer future alternative therapy for chronic otitis media.

Panel 4: Recent advances in otitis media in molecular biology, biochemistry, genetics, and animal models.

BACKGROUND: Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. OBJECTIVE: To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas. DATA SOURCES AND REVIEW METHODS: A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. RESULTS: Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.

A mouse with an N-Ethyl-N-nitrosourea (ENU) Induced Trp589Arg Galnt3 mutation represents a model for hyperphosphataemic familial tumoural calcinosis.

Mutations of UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetyl galactosaminyl transferase 3 (GALNT3) result in familial tumoural calcinosis (FTC) and the hyperostosis-hyperphosphataemia syndrome (HHS), which are autosomal recessive disorders characterised by soft-tissue calcification and hyperphosphataemia. To facilitate in vivo studies of these heritable disorders of phosphate homeostasis, we embarked on establishing a mouse model by assessing progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), and identified a mutant mouse, TCAL, with autosomal recessive inheritance of ectopic calcification, which involved multiple tissues, and hyperphosphataemia; the phenotype was designated TCAL and the locus, Tcal. TCAL males were infertile with loss of Sertoli cells and spermatozoa, and increased testicular apoptosis. Genetic mapping localized Tcal to chromosome 2 (62.64-71.11 Mb) which contained the Galnt3. DNA sequence analysis identified a Galnt3 missense mutation (Trp589Arg) in TCAL mice. Transient transfection of wild-type and mutant Galnt3-enhanced green fluorescent protein (EGFP) constructs in COS-7 cells revealed endoplasmic reticulum retention of the Trp589Arg mutant and Western blot analysis of kidney homogenates demonstrated defective glycosylation of Galnt3 in Tcal/Tcal mice. Tcal/Tcal mice had normal plasma calcium and parathyroid hormone concentrations; decreased alkaline phosphatase activity and intact Fgf23 concentrations; and elevation of circulating 1,25-dihydroxyvitamin D. Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that Tcal/Tcal mice had increased expression of Galnt3 and Fgf23 in bone, but that renal expression of Klotho, 25-hydroxyvitamin D-1α-hydroxylase (Cyp27b1), and the sodium-phosphate co-transporters type-IIa and -IIc was similar to that in wild-type mice. Thus, TCAL mice have the phenotypic features of FTC and HHS, and provide a model for these disorders of phosphate metabolism.

EVI1 acts as an inducible negative-feedback regulator of NF-κB by inhibiting p65 acetylation.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB-dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

A mouse model for osseous heteroplasia.

GNAS/Gnas encodes G(s)α that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G(s)α. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G(s)α. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G(s)α deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G(s)α and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G(s)α signalling pathways play a vital role in repressing ectopic bone formation.

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation.

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Oxr1 is essential for protection against oxidative stress-induced neurodegeneration.

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease.

Oto-endoscopy: a reliable and validated technique for phenotyping otitis media in the mouse.

The mouse is a widely used model for investigating the pathophysiological and genetic bases of otitis media (OM). It has proven a valuable tool for investigating the multifactorial bases of OM including the role of pathogens, anatomical factors, inflammatory mediators and susceptibility loci. However, straightforward and robust phenotyping tools for identifying murine otitis media are lacking, which has precluded for example the identification of mice with OM in genetic screens without resorting to time-consuming histopathology. We have set out to develop a phenotyping platform for the detection of OM in mice utilizing oto-endoscopy. We have applied the technique to a cohort of mice genetically susceptible to chronic otitis media. We show that oto-endoscopy is a safe, reliable and valid method for detecting otitis media in the mouse and discuss its utility in screens to identify novel genes involved with susceptibility to OM.

Multiple endocrine neoplasia type 1 knockout mice develop parathyroid, pancreatic, pituitary and adrenal tumours with hypercalcaemia, hypophosphataemia and hypercorticosteronaemia.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized in man by parathyroid, pancreatic, pituitary and adrenal tumours. The MEN1 gene encodes a 610-amino acid protein (menin) which is a tumour suppressor. To investigate the in vivo role of menin, we developed a mouse model, by deleting Men1 exons 1 and 2 and investigated this for MEN1-associated tumours and serum abnormalities. Men1(+/-) mice were viable and fertile, and 220 Men1(+/-) and 94 Men1(+/+) mice were studied between the ages of 3 and 21 months. Survival in Men1(+/-) mice was significantly lower than in Men1(+/+) mice (<68% vs >85%, P<0.01). Men1(+/-) mice developed, by 9 months of age, parathyroid hyperplasia, pancreatic tumours which were mostly insulinomas, by 12 months of age, pituitary tumours which were mostly prolactinomas, and by 15 months parathyroid adenomas and adrenal cortical tumours. Loss of heterozygosity and menin expression was demonstrated in the tumours, consistent with a tumour suppressor role for the Men1 gene. Men1(+/-) mice with parathyroid neoplasms were hypercalcaemic and hypophosphataemic, with inappropriately normal serum parathyroid hormone concentrations. Pancreatic and pituitary tumours expressed chromogranin A (CgA), somatostatin receptor type 2 and vascular endothelial growth factor-A. Serum CgA concentrations in Men1(+/-) mice were not elevated. Adrenocortical tumours, which immunostained for 3-beta-hydroxysteroid dehydrogenase, developed in seven Men1(+/-) mice, but resulted in hypercorticosteronaemia in one out of the four mice that were investigated. Thus, these Men1(+/-) mice are representative of MEN1 in man, and will help in investigating molecular mechanisms and treatments for endocrine tumours.

Spontaneous exocrine pancreas hypoplasia in specific pathogen-free C3HeB/FeJ and 101/H mouse pups causes steatorrhea and runting.

Under specific pathogen-free conditions, 1.3% to 1.8% of litters born in our inbred 101/H and C3HeB/FeJ mouse colonies had pups with steatorrhea and runting. Clinically affected male and female pups were first identified when they were from 14 to 25 d old. Unaffected littermates were healthy and were weaned successfully. Postmortem findings in 8 clinically affected mice included a small, poorly differentiated exocrine pancreas comprising cytokeratin-negative duct-like structures but lacking recognizable acinar cells with their normal carboxypeptidase B-positive zymogen granules. Endocrine pancreas islets were unremarkable and contained insulin-positive beta cells and glucagon-positive alpha cells. There was mild inflammation of the hindgut but no evidence of intestinal pathogens or marked inflammation or necrosis of pancreas, either alone or as part of a multisystemic inflammatory condition. Sera from pups in 4 affected litters did not contain antibodies to reovirus 3, mouse coronavirus, rotavirus, or mouse adenovirus 2. Furthermore, 4 sets of parental mice and sentinel mice from the facility were negative for 13 viruses, bacteria, and parasites. C3HeB/FeJ and 101/H inbred strains may be genetically predisposed because the steatorrhea and runting was absent in 13 other mouse strains and subspecies bred in the specific pathogen-free facility. This condition resembles exocrine pancreas hypoplasia, but the inheritance is complex. A wider implication is that runting coupled with steatorrhea are phenotypic criteria to suspect pancreatic disease that could be used in the context of a mouse N-ethyl-N-nitrosourea-mutagenesis program to identify potential mutants with defects in pancreas development.

Mutation at the Evi1 locus in Junbo mice causes susceptibility to otitis media.

Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.