Disruption Of Cochlear Development By Murine Cytomegalovirus Infection And Therapeutic Approaches That Prevent Hearing Loss

Authors

• Matthew Smith, University of Alabama at Birmingham

Advisor(s)

William Britt

Committee Member(s)

Chander Raman
Lynn Drobunz
Paul Goepfert
William Swords

School

Joint Health Sciences (Interdisciplinary)

Document Type

Dissertation

Department (new version)

Microbiology

Date of Award

9-9-2024

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous virus, infecting greater than 50% of most populations worldwide. Although rarely causing symptomatic infection in immunocompetent individuals, HCMV can cause life-threatening and severe end-organ disease in immunocompromised hosts including the developing fetus. HCMV can cross the placenta and is the most common viral infection of the developing fetus. Most newborn infants with congenital HCMV infection (cCMV) exhibit no clinical evidence of infection; however, up to 15% will develop long term neurodevelopmental sequelae including sensorineural hearing loss (SNHL). No prophylactic vaccines or effective antiviral treatments exist for cCMV infection, which may in part be secondary to the current gap in our understanding of the mechanisms by which HCMV leads to SNHL. Several models exist to study cCMV infection, and each has their advantages and disadvantages. It is well known that mice, deaf at birth, undergo significant central nervous system (CNS) and auditory development postnatally. We have employed a murine model of infection with murine CMV (MCMV) during the immediate postnatal period and subsequent analysis of the CNS and auditory development to further define the impact of viral infection on neurodevelopment. Intraperitoneal injection of mice within 24 hours of birth leads to robust viral replication in most organs with hematogenous dissemination to the cochlea and the brain. By infecting mice at PND0, we can establish an MCMV infection at a comparable period of development of the inner ear as a human infant infected in utero during the mid-2nd trimester of gestation. We have shown using this model of cCMV infection that MCMV induces a robust inflammatory response in the cochlea and have provided evidence that this inflammatory response may be playing a more important role in inner ear pathology than direct viral cytopathology. Combining two MCMV specific monoclonal antibodies (mAb) that have been shown to be active in vivo, we designed a treatment protocol to determine the impact of anti-MCMV antibodies on the viral load in the cochlea of infected mice, the expression of inflammatory mediators in the cochlea, and the downstream development of a disease phenotype, sensorineural hearing loss. Intraperitoneal injection of mAbs was given at PND2 and PND5 as early treatment was determined to be effective in controlling virus replication and disease in pilot experiments. We found that mAb treatment significantly decreased the viral load in the cochlea and blood over the course of acute infection at PND4, 8, and 14, but did not eliminate virus from organs of interest at any timepoint. Nonetheless, mAb treatment decreased the expression of the key inflammatory mediators IFIT1, TNFα, IFNγ, and IL1β at all given timepoints and reduced the incidence of hearing loss and histopathological changes in the cochlea, i.e., spiral ganglion neuron loss. These findings argue that sterilizing immunity may not be an absolute requirement to prevent damage to the developing auditory system and SNHL, but rather that the virusinduced cochlear inflammation may be playing a larger role in pathological outcomes such as hearing loss and cellular death. To further define the role of virus-induced inflammation in SNHL that follows MCMV infection of newborn mice, we performed bulk-RNA seq on cochleae of infected and uninfected mice aged PND4. We found by pathway analysis that many GO terms related to tissue remodeling, antiviral response, and immune response were significantly dysregulated. Further analysis of the differentially expressed genes (DEG) allowed us to determine the impact of MCMV infection on genes known to be related to hearing loss (DRG). Mutations in these genes have been found in humans and shown to lead to SNHL. We found that many of these DRGs are also DEGs already by PND4. This finding provides evidence that MCMV infection and virus-induced inflammation is altering the expression of critical host genes required for the development of hearing during postnatal maturation of auditory function in mice. The dysregulation of expression of these DRGs as early as PND4 combined with the importance of early mAb treatment at PND2 argues that critical auditory pathways are developing very early in the neonatal mouse and that treatment with antiviral antibodies can alter both the kinetics and the magnitude of cochlear infection, ultimately modulating virus-induced inflammation. Limiting virus-induced inflammation during this critical period of auditory development can in turn lessen the likelihood of SNHL in infected mice treated with antiviral antibodies. In a second series of studies utilizing this model, we investigated the impact of MCMV infection on the stria vascularis (SV), a structure in the lateral wall of the cochlea which is critical for the normal function of the Organ of Corti and hearing. The SV is a 3- cell layered specialized epithelium that maintains the unique biological fluid that bathes the stereocilia on the apical pole of hair cells, the highly electrically positive, potassium (K+) enriched (150mM) endocochlear fluid. The SV is also electrically isolated by tight junction barriers in both outer cell layers that create an impermeable extracellular barrier. Within the epithelium, K+ is transported to each cell type and finally out into the endolymph. We found that MCMV infected mice displayed altered gene and protein expression of multiple tight junction, gap junction, and ion channels necessary for normal SV function, leading to increased permeability within the SV. Additionally, the barrier surrounding the vasculature which is maintained by macrophages and pericytes was also found to have been compromised during MCMV infection. Increased SV permeability has been shown to lead to SNHL secondary to a failure to maintain the electrical potential differences in hair cells and the endolymph. These findings suggest that MCMV infection during development of the cochlea leads to decreased function(s) of the SV, a possible mechanism that could contribute to MCMV induced hearing loss and by analogy to SNHL following intrauterine HCMV infection. Finally, we found that treatment of MCMV infected mice with an anti-inflammatory glucocorticoid effectively decreased the inflammatory environment in the cochlea without altering the viral load. In addition, glucocorticoid treatment prevented decreased dysregulation of genes in resident cell of the SV and resulted in significantly lower incidence of SNHL in MCMV infected mice. These findings support a growing pool of evidence that the proinflammatory environment of the cochlea in the context of MCMV infection is directly responsible for the development of SNHL and that viral cytopathology has a minor role in SNHL in this model of hearing loss associated with cCMV infection.

ProQuest ID

https://proquest.com/docview/3101909438