Alternate Splicing of Interleukin-1 Receptor Type II (IL1R2) In Vitro Correlates with Clinical Glucocorticoid Responsiveness in Patients with AIED

Autoimmune Inner Ear Disease (AIED) is poorly characterized clinically, with no definitive laboratory test. All patients suspected of having AIED are given glucocorticoids during periods of acute hearing loss, however, only half initially respond, and still fewer respond over time

Microarray analysis of human keratinocytes from different anatomic sites reveals site-specific immune signaling and responses to human papillomavirus type 16 transfection

Abstract Background Stratified human keratinocytes (SHKs) are an essential part of mucosal innate immune response that modulates adaptive immunity to microbes encountered in the environment. The importance of these SHKs in mucosal integrity and development has been well characterized, however their regulatory immunologic role at different mucosal sites, has not. In this study we compared the immune gene expression of SHKs from five different anatomical sites before and after HPV16 transfection using microarray analyses. Methods Individual pools of human keratinocytes from foreskin, cervix, vagina, gingiva, and tonsils (HFKs, HCKs, HVKs, HGKs and HTLKs) were prepared. Organotypic (raft) cultures were established for both normal and HPV16 immortalized HFKs, HCKs, HVKs, HGKs and HTLKs lines which stably maintained episomal HPV16 DNA. Microarray analysis was carried out using the HumanHT-12 V4 gene chip (Illumina). Immune gene expression profiles were obtained by global gene chip (GeneSifter) and Ingenuity pathway analysis (IPA) for each individual site, with or without HPV16 transfection. Results We examined site specific innate immune response gene expression in SHKs from all five different anatomical sites before and after HPV16 transfection. We observed marked differences in SHK immune gene repertoires within and between mucosal tracts before HPV 16 infection. In addition, we observed additional changes in SHKs immune gene repertoire patterns when these SHKs were productively transfected with HPV16. Some immune response genes were similarly expressed by SHKs from different sites. However, there was also variable expression of non-immune response genes, such as keratin genes, by the different SHKs. Conclusions Our results suggest that keratinocytes from different anatomical sites are likely hard wired in their innate immune responses, and that these immune responses are unique depending on the anatomical site from which the SHKs were derived. These observations may help explain why select HPV types predominate at different mucosal sites, cause persistent infection at these sites, and on occasion, lead to HPV induced malignant and benign tumor development

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells

Immune Dysregulation in Patients Persistently Infected with Human Papillomaviruses 6 and 11

Human Papillomaviruses (HPVs) 6 and 11 are part of a large family of small DNA viruses, some of which are commensal. Although much of the population can contain or clear infection with these viruses, there is a subset of individuals who develop persistent infection that can cause significant morbidity and on occasion mortality. Depending on the site of infection, patients chronically infected with these viruses develop either recurrent, and on occasion, severe genital warts or recurrent respiratory papillomas that can obstruct the upper airway. The HPV-induced diseases described are likely the result of a complex and localized immune suppressive milieu that is characteristic of patients with persistent HPV infection. We review data that documents impaired Langerhans cell responses and maturation, describes the polarized adaptive T-cell immune responses made to these viruses, and the expression of class select II MHC and KIR genes that associate with severe HPV6 and 11 induced disease. Finally, we review evidence that documents the polarization of functional TH2 and T-regulatory T-cells in tissues persistently infected with HPV6 and 11, and we review evidence that there is suppression of natural killer cell function. Together, these altered innate and adaptive immune responses contribute to the cellular and humoral microenvironment that supports HPV 6 and 11-induced disease

Clinical History of patients treated with prednisone.

<p>“Prior tx?”: although all had prior rapid declines in hearing requiring prednisone therapy, none were treated in a 3 month period prior to enrollment. Several patients had occasional vestibular symptoms, however, these symptoms did not coincide with periods of hearing fluctuation. Serology was performed at Immco Diagnostics. NA = not available: serologic testing at Immco could not be performed as these tests were not covered b y the patient's insurance carrier. Audiometric change shown represents the change between the average of the post-treatment pure tone thresholds from the pre-treatment pure tone average threshold. NS = not significant: average post-treatment hearing changes of less than 5 dB are not felt to represent significant changes. All declines in hearing are reported as 0 dB. NR = clinical non-responder; R = clinical responder.</p

Differential expression of IL1R2 by microarray in response to autologous cochlear perilymph in patients undergoing cochlear implantation.

<p>1A Comparison of RNA expression using the Affymetrix U133A 2.0 chip to compare cultured PBMC of controls (CTRL) and AIED patients for the following conditions: either unstimulated (UNTX), stimulated with 23-valent pneumococcal vaccine (PNEUMO) or autologous cochlear perilymph (C). Only 10 genes (panel A) were significantly different (p<0.05) when a threshold of 2 and a Benjamini & Hochberg correction was applied (Genesifter, VixXlabs). Only one of them unique to the cochlear fluid stimulated condition IL1R2 (affy ID 205403), P<0.05. Array data in 1A can be viewed at <a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">www.ncbi.nlm.nih.gov/geo/</a> using the user “Vambutas” with a password “daniella” to view series “GSE4277” containing 18 arrays from the six patients with “1” next to their description in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-t001" target="_blank">table 1</a>. 1B A comparative analysis of IL1R2 expression in these arrays is seen in panel 1B, with standard deviations shown.</p

Clinical History of AIED and control patients undergoing cochlear implantation.

<p>PTA: Pure tone average of the audiogram at 500, 1000, 2000 and 4000 Hz, (although 4000 Hz is not traditionally included, this information is important to speech perception for cochlear implantees and therefore PTA represents these four frequencies for these patients). NP: Not Performed: control subjects did not have serology for AIED performed as there was no clinical indication for such studies, HINT: Hearing in Noise Test; IT-MAIS: Infant-Toddler Meaningful Auditory Integration Scale, LVA: Large Vestibular Aqueduct; Cx26: connexin 26 gene mutation. Superscript numbers in column 1 refer to inclusion of the patient in data sets for the below figures: 1: included in analysis for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-g001" target="_blank">figure 1</a>; 2b & c: included in analysis for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-g002" target="_blank">figure 2b or 2c</a>. In one patient, all RNA was used for microarray analysis (79 y/o female, control).</p