Harold R. Neely

Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human

Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3343A allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

Phenotype and Genetics of Progressive Sensorineural Hearing Loss (Snhl1) in the LXS Set of Recombinant Inbred Strains of Mice

Progressive sensorineural hearing loss is the most common form of acquired hearing impairment in the human population. It is also highly prevalent in inbred strains of mice, providing an experimental avenue to systematically map genetic risk factors and to dissect the molecular pathways that orchestrate hearing in peripheral sensory hair cells. Therefore, we ascertained hearing function in the inbred long sleep (ILS) and inbred short sleep (ISS) strains. Using auditory-evoked brain stem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements, we found that ISS mice developed a high-frequency hearing loss at twelve weeks of age that progressed to lower frequencies by 26 weeks of age in the presence of normal endocochlear potentials and unremarkable inner ear histology. ILS mice exhibited milder hearing loss, showing elevated thresholds and reduced DPOAEs at the higher frequencies by 26 weeks of age. To map the genetic variants that underlie this hearing loss we computed ABR thresholds of 63 recombinant inbred stains derived from the ISS and ILS founder strains. A single locus was linked to markers associated with ISS alleles on chromosome 10 with a highly significant logarithm of odds (LOD) score of 15.8. The 2-LOD confidence interval spans ∼4 Megabases located at position 54–60 Mb. This locus, termed sensorineural hearing loss 1 (Snhl1), accounts for approximately 82% of the phenotypic variation. In summary, this study identifies a novel hearing loss locus on chromosome 10 and attests to the prevalence and genetic heterogeneity of progressive hearing loss in common mouse strains.

Evolution of Myeloid Cells

In 1882, Elie Metchnikoff identified myeloid-like cells from starfish larvae responding to the invasion by a foreign body (rose thorn). This marked the origins for the study of innate immunity, and an appreciation that cellular immunity was well established even in these primitive organisms. This chapter focuses on these myeloid cells as well as the newest members of this family, the dendritic cells, and explores their evolutionary origins. Our goal is to provide evolutionary context for the development of the multilayered immune system of mammals, where myeloid cells now serve as central effectors of innate immunity and regulators of adaptive immunity. Overall, we find that core contributions of myeloid cells to the regulation of inflammation are based on mechanisms that have been honed over hundreds of millions of years of evolution. Using phagocytosis as a platform, we show how fairly simple beginnings have offered a robust foundation onto which additional control features have been integrated, resulting in central regulatory nodes that now manage multifactorial aspects of homeostasis and immunity.

Polygenic inheritance of sensorineural hearing loss (Snhl2, -3, and -4) and organ of Corti patterning defect in the ALR/LtJ mouse strain.

Progressive sensorineural hearing loss in humans is a common and debilitating impairment. Sensorineural deafness in inbred strains of mice is a similarly common and genetically diverse phenotype providing experimental models to study the underlying genetics and the biological effects of the risk factors. Here, we report that ALR/LtJ mice develop early-onset profound sensorineural hearing loss as evidenced by high-to-low frequency hearing threshold shifts, absent distortion-product otoacoustic emissions, and normal endocochlear potentials. Linkage analyses of a segregating backcross revealed three novel quantitative trait loci named sensorineural hearing loss (Snhl) -2, -3, and -4. The QTLs achieved very high LOD scores with markers on chromosome 1 (Snhl2, LOD: 12), chromosome 6 (Snhl3, LOD: 24) and chromosome 10 (Snhl4, LOD: 11). Together, they explained 90% of the phenotypic variance. While Snhl2 and Snhl3 affected hearing thresholds across a broad range of test frequencies, Snhl4 caused primarily high-frequency hearing loss. The hearing impairment is accompanied by an organ of Corti patterning defect that is characterized by the ectopic expression of supernumerary outer hair cells organized in rows along the abneural site of the sensory epithelium in the presence of unaltered planar polarity and otherwise normal cochlear duct morphology. Cloning the Snhl2, -3, and -4 genes in the ALR/LtJ mice may provide important genetic and mechanistic insights into the pathology of human progressive sensorineural deafness.

High-Frequency Sensorineural Hearing Loss and Its Underlying Genetics (Hfhl1 and Hfhl2) in NIH Swiss Mice

Studies using inbred strains of mice have been invaluable for identifying alleles that adversely affect hearing. However, the efficacy of those studies is limited by the phenotypes that these strains express and the alleles that they segregate. Here, by selectively breeding phenotypically and genetically heterogeneous NIH Swiss mice, we generated two lines—the all-frequency hearing loss (AFHL) line and the high-frequency hearing loss (HFHL) line—with differential hearing loss. The AFHL line exhibited characteristics typical of severe, early-onset, sensorineural hearing impairment. In contrast, the HFHL line expressed a novel early-onset, mildly progressive, and frequency-specific sensorineural hearing loss. By quantitative trait loci (QTLs) analyses in these two lines, we identified QTLs on chromosomes 7, 8, and 10 that significantly affected hearing function. The loci on chromosomes 7 and 8 (Hfhl1 and Hfhl2, respectively) are novel and appear to adversely affect only high frequencies (≥30xa0kHz). Mice homozygous for NIH Swiss alleles at either Hfhl1 or Hfhl2 have 32-kHz auditory-evoked brain stem response thresholds that are 8–14xa0dB SPL higher than the corresponding heterozygotes. DNA sequence analyses suggest that both the Cdh23ahl and Gipc3ahl5 variants contribute to the chromosome 10 QTL detected in the AFHL line. The frequency-specific hearing loss indicates that the Hfhl1 and Hfhl2 alleles may affect tonotopic development. In addition, dissecting the underlying complex genetics of high-frequency hearing loss may prove relevant in identifying less severe and common forms of hearing impairment in the human population.

Rapid Induction of Splenic and Peritoneal B-1a Cells in Adult Mice by Thymus-Independent Type-2 Antigen

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.

Depletion of B220+NK1.1+ cells enhances the rejection of established melanoma by tumor-specific CD4+ T cells

Five-year survival rates for patients diagnosed with metastatic melanoma are less than 5%. Adoptive cell transfer (ACT) has achieved an objective response of 50% by Response Evaluation Criteria in Solid Tumors (RECIST) in this patient population. For ACT to be maximally effective, the host must first be lymphodepleted. It is hypothesized that lymphodepletion may remove regulatory elements and cytokine sinks, or increase the activation and availability of antigen presenting cells (APCs). We use an in vivo model to study the ACT of tumor-associated antigen (TAA)-specific CD4+ T cells (TRP-1 cells). We have discovered that depletion of NK1.1+ cells enhances the rejection of established melanoma tumors by adoptively transferred TRP-1 CD4+ T cells. NK1.1+ cell depletion increases the number of CD4+ T cells, the serum concentration of pro-inflammatory cytokines, autoimmune vitiligo, host survival and prevented recurrence after ACT. Because multiple cells express NK1.1, we targeted different NK1.1+ cell populations using antibodies specific for NK cells, pre-mNK cells, and innate lymphoid cells (ILCs). Our data suggests that NK1.1+B220+ pre-mNK cells (also known as interferon-producing killer dendritic cells; IKDCs) are an important inhibitor of the CD4+ T cell response to melanoma. Understanding this mechanism may help design new immunotherapies to modulate the activity of pre-mNKs in the face of an antitumor immune response and inhibit their suppression of adoptively transferred T cells.

CXCL13 Responsiveness but Not CXCR5 Expression by Late Transitional B Cells Initiates Splenic White Pulp Formation

Secondary lymphoid organs (SLO) provide the structural framework for coconcentration of Ag and Ag-specific lymphocytes required for an efficient adaptive immune system. The spleen is the primordial SLO, and evolved concurrently with Ig/TCR:pMHC-based adaptive immunity. The earliest cellular/histological event in the ontogeny of the spleen’s lymphoid architecture, the white pulp (WP), is the accumulation of B cells around splenic vasculature, an evolutionarily conserved feature since the spleen’s emergence in early jawed vertebrates such as sharks. In mammals, B cells are indispensable for both formation and maintenance of SLO microarchitecture; their expression of lymphotoxin α1β2 (LTα1β2) is required for the LTα1β2:CXCL13 positive feedback loop without which SLO cannot properly form. Despite the spleen’s central role in the evolution of adaptive immunity, neither the initiating event nor the B cell subset necessary for WP formation has been identified. We therefore sought to identify both in mouse. We detected CXCL13 protein in late embryonic splenic vasculature, and its expression was TNF-α and RAG-2 independent. A substantial influx of CXCR5+ transitional B cells into the spleen occurred 18 h before birth. However, these late embryonic B cells were unresponsive to CXCL13 (although responsive to CXCL12) and phenotypically indistinguishable from blood-derived B cells. Only after birth did B cells acquire CXCL13 responsiveness, accumulate around splenic vasculature, and establish the uniquely splenic B cell compartment, enriched for CXCL13-responsive late transitional cells. Thus, CXCL13 is the initiating component of the CXCL13:LTα1β2 positive feedback loop required for WP ontogeny, and CXCL13-responsive late transitional B cells are the initiating subset.

Normal hearing in α-galactosidase A-deficient mice, the mouse model for Fabry disease

Abstract Fabry disease (OMIM 301500) is a rare X-linked recessive disorder caused by mutations in the α-galactosidase gene (Gla). Loss of Gla activity leads to the abnormal accumulation of glycosphingolipids in lysosomes of predominantly vascular endothelial cells. Clinically the disorder presents with angiokeratomas, clouding of the cornea, and renal, cardiac, and cerebrovascular complications. In addition, there is an increased incidence of sensorineural hearing loss in Fabry patients. In this study, we investigated the loss of α-galactosidase A activity on hearing function in Gla-deficient mice (Glatm1Kul). Gla mRNA was readily detected in the cochlea of 2- and 12-month old C57BL/6J and C3HeB/FeJ mice. The targeted allele was introgressed to the normal hearing C3HeB/FeJ strain to eliminate confounding genetic background effects. Auditory brain stem responses (ABR) to click, 8-, 16-, and 32 kHz stimuli measured at regular intervals from animals at the N4 backcross generation and from N4F1 hybrids demonstrated normal hearing in hemizygous and homozygous mutant mice up to 76 weeks of age. By histological criteria, the cyto-architecture of the mutant cochlea showed a normal appearance. The data demonstrate that in the mouse the loss of α-galactosidase A activity is genetically or biochemically buffered and not sufficient per se to cause an appreciable degree of hearing impairment.

“Double-duty” conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC‐dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.