Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4+ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4+ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4+ and CD8+ T cells, persistent expansion of all circulating CD8+ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4+ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection

A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82 % correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0722-x) contains supplementary material, which is available to authorized users

Ageing-Induced Decline in Primary Myeloid Cell Phagocytosis Is Unaffected by Optineurin Insufficiency

Optineurin is a ubiquitin-binding adaptor protein involved in multiple cellular processes, including innate inflammatory signalling. Mutations in optineurin were found in amyotrophic lateral sclerosis, an adult-onset fatal neurodegenerative disease that targets motor neurons. Neurodegeneration results in generation of neuronal debris, which is primarily cleared by myeloid cells. To assess the role of optineurin in phagocytosis, we performed a flow cytometry-based phagocytic assay of apoptotic neuronal debris and E. coli bioparticles in bone marrow-derived macrophages (BMDMs), and primary neonatal microglia from wild-type (WT) and optineurin-insufficient (Optn470T) mice. We found no difference in phagocytosis efficiency and the accompanying cytokine secretion in WT and Optn470T BMDMs and microglia. This was true at both steady state and upon proinflammatory polarization with lipopolysaccharide. When we analysed the effect of ageing as a major risk factor for neurodegeneration, we found a substantial decrease in the percentage of phagocytic cells and proinflammatory cytokine secretion in BMDMs from 2-year-old mice. However, this ageing-induced phagocytic decline was unaffected by optineurin insufficiency. All together, these results indicate that ageing is the factor that perturbs normal phagocytosis and proinflammatory cytokine secretion, but that optineurin is dispensable for these processes

Distinct gene alterations between Fos-expressing striatal and thalamic neurons after withdrawal from methamphetamine self-administration

Background Methamphetamine (Meth) seeking progressively increases after withdrawal (incubation of Meth craving). We previously demonstrated a role of anterior intralaminar nucleus of thalamus (AIT) to dorsomedial striatum (DMS) projections in this incubation. Here, we examined molecular alterations in DMS and AIT neurons activated (identified by neuronal activity marker Fos) during &quot;incubated&quot; Meth-seeking relapse test after prolonged withdrawal. Methods We trained male rats to self-administer Meth or saline (control condition) for 10 days (6 hr/day). Using fluorescence-activated cell sorting, we examined gene expression in Fos-positive (activated during a 2-hr relapse test) and Fos-negative (nonactivated) DMS and AIT neurons. Results In DMS, we found increased mRNA expressions of immediate early genes (IEGs) (Arc, Egr1, Npas4, Fosb), Trkb, glutamate receptors subunits (Gria3, Grin1, Grin2b, Grm1), and epigenetic enzymes (Hdac3, Hdac5, Crebbp) in Fos-positive neurons, compared with Fos-negative neurons. In AIT, we found that fewer genes (Egr1, Fosb, TrkB, Grin1, and Hdac5) exhibited increased mRNA expression in Fos-positive neurons. Unexpectedly, in both brain regions, gene alterations described above also occurred in drug-naive saline self-administration control rats. Conclusions These results demonstrated that transcriptional regulations in Fos-positive neurons activated during the relapse tests are brain region-specific but are not uniquely associated with drug exposure during the self-administration training.Background Methamphetamine (Meth) seeking progressively increases after withdrawal (incubation of Meth craving). We previously demonstrated a role of anterior intralaminar nucleus of thalamus (AIT) to dorsomedial striatum (DMS) projections in this incubation. Here, we examined molecular alterations in DMS and AIT neurons activated (identified by neuronal activity marker Fos) during &quot;incubated&quot; Meth-seeking relapse test after prolonged withdrawal. Methods We trained male rats to self-administer Meth or saline (control condition) for 10 days (6 hr/day). Using fluorescence-activated cell sorting, we examined gene expression in Fos-positive (activated during a 2-hr relapse test) and Fos-negative (nonactivated) DMS and AIT neurons. Results In DMS, we found increased mRNA expressions of immediate early genes (IEGs) (Arc, Egr1, Npas4, Fosb), Trkb, glutamate receptors subunits (Gria3, Grin1, Grin2b, Grm1), and epigenetic enzymes (Hdac3, Hdac5, Crebbp) in Fos-positive neurons, compared with Fos-negative neurons. In AIT, we found that fewer genes (Egr1, Fosb, TrkB, Grin1, and Hdac5) exhibited increased mRNA expression in Fos-positive neurons. Unexpectedly, in both brain regions, gene alterations described above also occurred in drug-naive saline self-administration control rats. Conclusions These results demonstrated that transcriptional regulations in Fos-positive neurons activated during the relapse tests are brain region-specific but are not uniquely associated with drug exposure during the self-administration training.</p

The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1.

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1

Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and β1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/β1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC50 range: 104–640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.—Lusso, P., Vangelista, L., Cimbro, R., Secchi, M., Sironi, F., Longhi, R., Faiella, M., Maglio, O., Pavone, V. Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity

Neuroimmune characterization of optineurin insufficiency mouse model during ageing

Optineurin is a multifunctional polyubiquitin-binding protein implicated in infammatory signalling. Optineurin mutations are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerative diseases characterised by neuronal loss, neuroinfammation, and peripheral immune disbalance. However, the pathogenic role of optineurin mutations is unclear. We previously observed no phenotype in the unmanipulated young optineurin insufciency mice (Optn470T), designed to mimic ALS/FTD-linked truncations defcient in polyubiquitin binding. The purpose of this study was to investigate whether ageing would trigger neurodegeneration. We performed a neurological, neuropathological, and immunological characterization of ageing wild-type (WT) and Optn470T mice. No motor or cognitive diferences were detected between the genotypes. Neuropathological analyses demonstrated signs of ageing including lipofuscin accumulation and microglial activation in WT mice. However, this was not worsened in Optn470T mice, and they did not exhibit TAR DNA-binding protein 43 (TDP-43) aggregation or neuronal loss. Spleen immunophenotyping uncovered T cell immunosenescence at two years but without notable diferences between the WT and Optn470T mice. Conventional dendritic cells (cDC) and macrophages exhibited increased expression of activation markers in two-year-old Optn470T males but not females, although the numbers of innate immune cells were similar between genotypes. Altogether, a combination of optineurin insufciency and ageing did not induce ALS/FTD-like immune imbalance and neuropathology in mice