Single-Cell Transcriptomes Reveal a Complex Cellular Landscape in the Middle Ear and Differential Capacities for Acute Response to Infection.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation

A nuclear localization signal at the SAM-SAM domain interface of AIDA-1 suggests a requirement for domain uncoupling prior to nuclear import

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus

Peptides actively transported across the tympanic membrane: Functional and structural properties.

Otitis media (OM) is the most common infectious disease of children under six, causing more antibiotic prescriptions and surgical procedures than any other pediatric condition. By screening a bacteriophage (phage) library genetically engineered to express random peptides on their surfaces, we discovered unique peptides that actively transport phage particles across the intact tympanic membrane (TM) and into the middle ear (ME). Herein our goals were to characterize the physiochemical peptide features that may underlie trans-TM phage transport; assess morphological and functional effects of phage peptides on the ME and inner ear (IE); and determine whether peptide-bearing phage transmigrate from the ME into the IE. Incubation of five peptide-bearing phage on the TM for over 4hrs resulted in demonstrably superior transport of one peptide, in level and in exponential increase over time. This suggests a preferred peptide motif for TM active transport. Functional and structural comparisons revealed unique features of this peptide: These include a central lysine residue, isoelectric point of 0.0 at physiological pH and a hydrophobic C-terminus. When the optimal peptide was applied to the TM independent of phage, similar transport was observed, indicating that integration into phage is not required. When 109 particles of the four different trans-TM phage were applied directly into the ME, no morphological effects were detected in the ME or IE when compared to saline or wild-type (WT) phage controls. Comparable, reversible hearing loss was observed for saline controls, WT phage and trans-TM peptide phage, suggesting a mild conductive hearing loss due to ME fluid. Perilymph titers after ME incubation established that few copies of trans-TM peptide phage crossed into the IE. The results suggest that, within the parameters tested, trans-TM peptides are safe and could be used as potential agents for noninvasive delivery of drugs, particles and gene therapy vectors to the ME

Single-Cell Transcriptomes Reveal a Complex Cellular Landscape in the Middle Ear and Differential Capacities for Acute Response to Infection.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens

Histological evaluation of the ear after trans-TM peptide phage application, compared to WT phage negative control and saline control.

<p><b>A.</b> Hematoxylin and eosin (H&E) stained sections illustrate the external auditory canal (EAC), TM and ME, 24 hrs after application of 10¹⁰ TM-3 phage or controls to the external surface of the TM. <b>B-E.</b> The ME and ME mucosa (<b>B</b>); the organ of Corti (C); the ME, RWM and cochlear basal turn (<b>D</b>) and the cochlea (<b>E</b>), 24 hrs after application of 10¹⁰ TM-3 phage directly into the ME.</p

Impact of trans-TM peptide phage on hearing.

<p>ABR thresholds measured in dB SPL (Sound Pressure Level relative to .0002 dynes/cm²) were recorded pre-operatively and daily for four days following 10¹⁰ phage particle injection into the ME bulla. Responses were obtained for 8, 16, and 32 kHz pure-tone stimuli. Results indicate that thresholds were elevated up to 20 dB on postoperative days 1 and 2, recovered to within +/- 5 dB of baseline by day 3. Results for four TM-transiting phage, WT phage and saline were similar, indicating that the temporary change in threshold is due to fluid, and that no damage to hearing occurred as a result of TM-transiting phage application. Data are means + SEM (n = 8).</p

Comparison of peptide phage transport to systemic antibiotic delivery in the ME.

<p><b>A.</b> Proportion of systemically delivered antibiotics that were observed in the ME of patients at 1, 2 or 3 hrs after systemic administration by Silverstein et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172158#pone.0172158.ref043" target="_blank">43</a>] (penicillin, 500,000 units; black bars) or Nicolau et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172158#pone.0172158.ref044" target="_blank">44</a>] (cefprozil, 15 mg/kg; white bars). <b>B.</b> Proportion of TM-3 peptide phage, applied to the TM, that was recovered from the ME 1, 2 or 4 hrs later, and TM-3 peptide, applied to the TM, recovered from the ME 1 hr later. Error bars = SEM (n = 4).</p

Kinetics of peptide-mediated trans-TM transport.

<p>ME recovery kinetics of five TM-transiting peptide phage, compared to WT phage particles, following incubation of 10¹⁰ phage on the exterior surface of the TM for 1, 2 or 4 hrs. <b>A</b> = log scale, <b>B</b> = linear scale. All TM-transiting peptide phage were recovered at levels much higher than were WT phage not bearing a peptide. Phage bearing TM-3 peptide were recovered at much higher titers (more than 10⁵ higher at 4 hrs) than WT phage, and significantly more than any other TM-transiting phage. TM-3 phage also exhibited exponential increases in ME recovery over time consistent with an active transport mechanism. Error bars = SEM (n = 6 animals).</p

Comparison of trans-TM peptide entry into the IE versus the ME.

<p>Compared to ME recovery of TM-3 after 4-hr application of 10⁹ phage to the TM (white bar, taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172158#pone.0172158.g001" target="_blank">Fig 1</a>), recovery of TM-transiting phage from perilymph, 4 hrs after instillation of 10⁹ phage onto the area of the RWM (black bars) is absent or very low. Phage recovery from the IE improved somewhat at 24 hrs, but was not greater for TM-transiting phage than for WT Phage not bearing a peptide. Thus, any movement of phage into the IE does not appear to be peptide-mediated. Error bars = SD (n = 4).</p