Viral Immune signatures from cerebrospinal fluid extracellular vesicles and particles in HAM and other chronic neurological diseases.

Background and objectives: Extracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease. Methods: We analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease. Results: Significant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without. Discussion: These data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals.

MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

A simple, high-throughput method of protein and label removal from extracellular vesicle samples.

Evidence continues to increase of the clinical utility extracellular vesicles (EVs) as translational biomarkers. While a wide variety of EV isolation and purification methods have been implemented, few techniques are high-throughput and scalable for removing excess fluorescent reagents (e.g. dyes, antibodies). EVs are too small to be recovered from routine cell-processing procedures, such as filtration or centrifugation. The lack of suitable methods for removing unbound labels, especially in optical assays, is a major roadblock to accurate EV phenotyping and utilization of EV assays in a translational or clinical setting. Therefore, we developed a method for using a multi-modal resin, referred to as EV-Clean, to remove unbound labels from EV samples, and we demonstrate improvement in flow cytometric EV analysis with the use of this EV-Clean method.

High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10-100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.

Spontaneous and influenza virus-induced sleep are altered in TNF-alpha double-receptor deficient mice.

Tumor necrosis factor-alpha (TNF-alpha) is associated with sleep regulation in health and disease. Previous studies assessed sleep in mice genetically deficient in the TNF-alpha 55-kDa receptor. In this study, spontaneous and influenza virus-induced sleep profiles were assessed in mice deficient in both the 55-kDa and 75-kDa TNF-alpha receptors [TNF-2R knockouts (KO)] and wild-type (WT) strain controls. Under baseline conditions the TNF-2R KO mice had less non-rapid eye movement sleep (NREMS) than WTs during the nighttime and more rapid eye movement sleep (REMS) than controls during the daytime. The differences between nighttime maximum and daytime minimum values of electroencephalogram (EEG) delta power during NREMS were greater in the TNF-2R KO mice than in WTs. Viral challenge (mouse-adapted influenza X-31) enhanced NREMS and decreased REMS in both strains roughly to the same extent. EEG delta power responses to viral challenge differed substantially between strains; the WT animals increased, whereas the TNF-2R KO mice decreased their EEG delta wave power during NREMS. There were no differences between strains in body temperatures or locomotor activity in uninfected mice or after viral challenge. Analyses of cortical mRNAs confirmed that the TNF-2R KO mice lacked both TNF-alpha receptors; these mice also had higher levels of orexin mRNA and reduced levels of the purine P2X7 receptor compared with WTs. Results reinforce the hypothesis that TNF-alpha is involved in physiological sleep regulation but plays a limited role in the acute-phase response induced by influenza virus.

Interferon type I receptor-deficient mice have altered disease symptoms in response to influenza virus.

The role of type I interferons (IFNs) in mediation of acute viral symptoms (fever, somnolence, anorexia, etc.) is unknown. To determine the role of type I IFN in selected symptom development, body temperature and sleep responses to a marginally lethal dose of X-31 influenza virus were examined in mice with a targeted mutation of the IFN receptor type I (IFN-RI knockouts) and compared to wild-type 129 SvEv control mice. Mice were monitored for 48 h to determine baseline temperature and sleep profiles prior to infection, and then for 9 days following infection. Hypothermic responses to virus were perceptible beginning at 64 h post-infection (PI) and were more marked in KO mice until 108 h, when hypothermia became more exaggerated in wild-type controls. Temperatures of wild-type mice continued to decline through day 9 while temperatures in IFN-RI KO mice stabilized. Time spent in non-rapid eye movement sleep (NREMS) increased in KO mice when hypothermia was marked and then returned to baseline levels, while NREMS continued to increase in wild-type mice through day 9. Other sleep parameters [time spent in rapid eye movement sleep (REMS), relative NREMS EEG slow wave activity, NREMS EEG power density] were all reduced in wild-type mice compared to KOs from days 3 to 8 while REMS low frequency EEG power density increased in wild-type relative to KOs. In conclusion, our results indicate that the presence of functional type I IFN slightly ameliorates disease symptoms early in the X-31 infection while exacerbating disease symptoms later in the infection.

Influenza virus-induced glucocorticoid and hypothalamic and lung cytokine mRNA responses in dwarf lit/lit mice.

Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.

Sleep and body temperature responses in an acute viral infection model are altered in interferon type I receptor-deficient mice.

Type I interferons (IFNs) include IFNalpha and IFNbeta, both of which are elevated in acute viral infections and both of which have been shown to induce symptoms such as fever and somnolence when administered in pharmacological doses. To investigate the role of type I IFNs in mediation of acute respiratory viral symptoms we examined sleep and body temperature responses in mice with a targeted mutation of the IFN receptor type I (IFN-RI knockouts). IFN-RI knockouts (KOs) or wild-type 129 SvEv controls were challenged intratracheally (IT) with combined poly[rI.rC] (synthetic double-stranded RNA) and IFNgamma, a model that simulates an acute viral infection with respect to body temperature and locomotor activity responses. Control mice of both strains were treated with IT IFNgamma alone. Hypothermic responses to IT poly[rI.rC]/IFNgamma were more exaggerated in the IFN-RI KO mice than in wild-type. The non-rapid eye movement sleep (NREMS) response to IT poly[rI.rC]/IFNgamma was increased earlier in the IFN-RI KO mice than in wild-type, though the total time spent in NREMS was reduced in the KOs compared to wild-type and the return to baseline NREMS was faster in the KOs. The quality of NREMS also was altered more extensively in the wild-type than in the KO mice. Spontaneous rapid eye movement sleep (REMS) was suppressed in IFN-RI KOs as previously reported, but was not substantially altered in either mouse strain by IT poly[rI.rC]/IFNgamma challenge. Our results implicate type I IFNs as inhibitors of the hypothermic response and enhancers of the NREMS response to IT poly[rI.rC]/IFNgamma, a model of acute viral infection.

Intratracheal double-stranded RNA plus interferon-gamma: a model for analysis of the acute phase response to respiratory viral infections.

Double-stranded (ds)RNA is made as a by-product of viral replication. Synthetic dsRNA induces virtually all of the same systemic symptoms as acute viral infections, such as fever and malaise. In order to develop a model of respiratory viral infections (such as influenza) suitable for use in gene knockout mice (where the deleted gene may affect viral replication), we examined C57BL/6 mouse body temperature and locomotor activity responses to the synthetic dsRNA polyriboinosinic.polyribocytidylic acid (poly[rI.rC]) introduced via the intratracheal (IT) route. We compared the IT poly[rI.rC] responses to the well-characterized intraperitoneal (IP) poly[rI.rC] responses. IT poly[rI.rC] failed to induce an acute phase response (APR) in mice, in contrast to IP poly[rI.rC]. However, addition of interferon (IFN)gamma to the IT poly[rI.rC] inoculum induced sustained hypothermia and suppressed locomotor activity responses with similar kinetics to those responses seen in acute mouse influenza. We further examined cytokine, antiviral, muscarinic M2 receptor and inducible nitric oxide synthase gene expression at 5 hr in the lungs of IT challenged mice. These studies suggested that priming the lung with IFNgamma could enhance proinflammatory (IL1beta, IL6, TNFalpha) cytokine gene expression and suppress interferon gene expression compared to IT poly[rI.rC] alone. No differences were detected for the other genes examined. While further molecular characterization of the model is required, we demonstrate that IT challenge with combined poly[rI.rC] and IFNgamma closely simulates the APR to an acute respiratory virus, and may serve as a suitable model for analyzing the molecular basis of the viral APR in gene knockout mice.

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity.

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.