Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67 phox , activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)-bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1

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

Analysis of the pH Dependence of the Neonatal Fc Receptor/Immunoglobulin G Interaction Using Antibody and Receptor Variants

The neonatal Fe receptor (FcRn) binds maternal immunoglobulin G (IgG) from ingested milk in the gut (pH 6.0-6.5) and delivers it to the bloodstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reproduces the physiological pH-dependent interaction with IgG, showing high-affinity binding at pH 6.0-6.5 but weak or no binding at pH 7.0-7.5. We have studied the pH dependence of the FcRn/IgG interaction using a surface plasmon resonance assay to measure kinetic and equilibrium constants. We show that the affinity of FcRn for IgG is reduced about 2 orders of magnitude as the pH is raised from 6.0 to 7.0. A Hill plot analysis suggests that several titrating residues participate in the pH-dependent affinity transition. Histidine side chains are likely candidates for residues that titrate between pH 6.0 and 7.0, and previous biochemical and structural work identified several histidines on the Fe portion of IgG that are located at the FcRn binding site. Using mutant IgG molecules and IgG subtype variants that differ in the number of histidines at the IgG/FcRn interface, we demonstrate that IgG histidines located at the junction between the CH2 and CH3 domains (residues 310 and 433) contribute to the pH-dependent affinity transition. Experiments with a mutant FcRn molecule show that two histidines on the FcRn heavy chain (residues 250 and 251) also contribute to the pH dependence of the FcRn/IgG interaction. These results are interpreted using the crystal structures of FcRn and an FcRn/Fc complex

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

Pre-treatment induced expression of mIL1R2 in response to dexamethasone correlates with post-treatment clinical hearing restoration.

<p>3A 18 patients with presumptive AIED and sudden declines in hearing were enrolled, blood drawn prior to treatment, and treated with 60 mg prednisone/day×7 days and then tapered. Pre-treatment PBMC were divided and incubated with either no stimulus or dexamethasone (8 µg/ml is shown). IL1R2 mRNA was measured by Q-RT-PCR. Post-treatment audiograms were obtained between 7–14 days later. Audiometric improvement (Δ) was measured as the average of 250, 500, 1,2,& 4 kHz. Responders (R) all returned to prior baseline hearing. Pre-treatment levels of mIL-1R2 Q-RT-PCR (expressed as fold change over baseline) predicted steroid responsiveness in AIED (P<0.0001, Mann Whitney test). 3B Pre and post treatment mIL1R2 levels in a subset of clinical responders and non-responders. Pre-treatment responders have no basal mIL1R2 expression in PBMC (unstim) at 45 cycles (in replicate samples), compared with non-responders that have substantially higher basal levels. Although PBMC from both responders and non-responders demonstrate increased mIL1R2 RNA expression in response to in vitro dexamethasone stimulation (dex stim), responders are exquisitely sensitive. The concentration of dexamethasone shown for the PBMC stimulation experiments was 20 ug/ml, however, 4 ug/ml and 8 ug/ml produced identical results, suggesting maximal stimulation even at 4 ug/ml. 3C Despite clear differences in mIL1R2 expression patterns, sIL1R2 expression is differences between responders and non-responders are minimal suggesting alternate splicing in clinical responders.</p