Alarmins and central nervous system inflammation in HIV-associated neurological disorders.

In the era of highly active antiretroviral therapy (HAART), HIV-1-associated neurocognitive disorders (HAND) persist in infected individuals with adequate immunological and virological status. Risk factors for cognitive impairment include hepatitis C virus co-infection, host genetic factors predisposing to HAND, the early establishment of the virus in the CNS and its persistence under HAART; thus, the CNS is an important reservoir for HIV. Microglial cells are permissive to HIV-1, and NLRP3 inflammasome-associated genes were found expressed in brains of HIV-1-infected persons, contributing to brain disease. Inflammasomes can be triggered by alarmins or danger-associated molecular patterns (DAMPs), which directly stimulate the production of proinflammatory mediators by glial cells, contribute to blood-brain barrier injury through induction of release of various proteases and allow the passage of infected macrophages, and trigger IL-1ß release from primed cells. Amongst alarmins involved in HIV-1-induced neuropathogenesis, IL-33 and high-mobility group box 1 (HMGB1) are of particular interest. Neurocognitive alterations were recently associated with dysregulation of the IL-33/ST2 axis in the CNS, leading to the induction of neuronal apoptosis, decrease in synaptic function and neuroinflammation. Specific biomarkers, including HMGB1 and anti-HMGB1 antibodies, have been identified in cerebrospinal fluid from patients with HAND, correlated with immune activation and identifying a very early stage of neurocognitive impairment that precedes changes in metabolites detected by magnetic resonance spectroscopy. Moreover, HMGB1 plays a crucial role in HIV-1 persistence in dendritic cells and in the constitution of viral reservoirs. In this review, the mechanisms whereby alarmins contribute to HIV-1-induced CNS inflammation and neuropathogenesis will be discussed.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Pitfalls in anti-influenza T cell detection by Elispot using thimerosal containing pandemic H1N1 vaccine as antigen.

Monitoring T cells in combination with humoral response may be of value to predict clinical protection and cross-protective immunity after influenza vaccination. Elispot technique which measures cytokine produced after antigen-specific T cell stimulation is used routinely to detect and characterize anti-viral T cells. We found that the preservative thimerosal present in most H1N1 pandemic vaccines, induced in vitro abortive activation of T cells followed by cell death leading to false-positive results with the Elispot technique. The size of the spots, usually not measured in routine analysis, appears to be a discriminative criterion to detect this bias. Multi-dose vials of vaccine containing thimerosal remain important for vaccine delivery and our results alert about false-positive results of Elispot to monitor the clinical efficacy of these vaccines. We showed that this finding extends for other T cell monitoring techniques based on cytokine production such as ELISA. Although measuring in vitro immune response using the whole vaccine used for human immunization directly reflects in vivo global host response to the vaccine, the present study strongly supports the use of individual vaccine components for immune monitoring due to the presence of contaminants, such as thimerosal, leading to a bias in interpretation of the results.

HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells.

Dendritic cells (DCs) initiate immune responses by transporting antigens and migrating to lymphoid tissues to initiate T-cell responses. DCs are located in the mucosal surfaces that are involved in human immunodeficiency virus (HIV) transmission and they are probably among the earliest targets of HIV-1 infection. DCs have an important role in viral transmission and dissemination, and HIV-1 has evolved different strategies to evade DC antiviral activity. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can act as an alarmin, a danger signal to alert the innate immune system for the initiation of host defense. It is the prototypic damage-associated molecular pattern molecule, and it can be secreted by innate cells, including DCs and natural killer (NK) cells. The fate of DCs is dependent on a cognate interaction with NK cells, which involves HMGB1 expressed at NK-DC synapse. HMGB1 is essential for DC maturation, migration to lymphoid tissues and functional type-1 polarization of naïve T cells. This review highlights the latest advances in our understanding of the impact of HIV on the interactions between HMGB1 and DCs, focusing on the mechanisms of HMGB1-dependent viral dissemination and persistence in DCs, and discussing the consequences on antiviral innate immunity, immune activation and HIV pathogenesis.

The suppression of immune activation during enfuvirtide-based salvage therapy is associated with reduced CCR5 expression and decreased concentrations of circulating interleukin-12 and IP-10 during 48 weeks of longitudinal follow-up.

BACKGROUND: It has been suggested that patients who initiate highly active antiretroviral therapy (HAART) late in their course of infection may have suboptimal CD4 T-cell gains, persistent alterations in T-cell subsets and residual inflammation. To address this issue, we carried out a comprehensive 48-week immunological study in HIV-infected patients who had experienced failures of prior therapies, had low CD4 cell counts, and were receiving enfuvirtide-based salvage therapy. METHODS: Immunological monitoring of peripheral lymphocytes from enfuvirtide-responder patients was performed over a 48-week period. A detailed assessment of immune cell subsets, their activation state [CD38 and human leucocyte antigen (HLA)-DR expression] and homeostasis [activation-induced cell death (AICD) and Ki67 expression], and the expression of co-receptors was performed by flow cytometry. Cytokine and chemokine signatures were assessed using multianalyte profiling technology. RESULTS: Enfuvirtide-based salvage therapy induced a progressive restoration of naïve and central memory CD4 T cells, associated with a decrease in their activation state, suppression of premature priming for AICD and increased expression of Ki67. In addition, a significant decrease in C-C chemokine receptor 5 (CCR5) expression was detected on CD4 T cells, which was strongly correlated with the suppression of immune activation. Changes in circulating proinflammatory molecules occurred; i.e. there were decreases in the concentrations of interleukin (IL)-12, macrophage inflammatory protein MIP-1α, MIP-1ß, monokine induced by IFNγ (MIG) and interferon-γ-inducible protein-10 (IP-10). The decline in circulating IL-12 and IP-10 was correlated with both the reduction in the viral load and CD4 T-cell restoration. CONCLUSIONS: This study shows that suppression of HIV-1 replication with enfuvirtide-based salvage therapy in patients with low CD4 cell counts may result in an immunological benefit, characterized by the restoration of CD4 T-cell subsets associated with decreased immune activation and suppression of inflammation.

Impact of gamma-chain cytokines on T cell homeostasis in HIV-1 infection: therapeutic implications.

CD4(+) T cell lymphocytes are a major target for human immunodeficiency virus type-1 (HIV-1) infection. During this chronic infection, CD4(+) T cell loss (induced through direct viral replication), generalized immune activation and increased susceptibility to apoptosis result in impaired T cell homeostasis with subsequent development of opportunistic infections and cancers. Highly active antiretroviral therapy (HAART) has a well-defined, beneficial effect on HIV-1-related clinical outcome; however, it does not lead to normalization of immune dysregulation. In order to boost both CD4(+) T cell restoration and HIV-1 specific immunity, immunotherapy with gamma-chain cytokines has been used in HIV-1-infected patients during concomitant HAART. In this review, we summarize the role of gamma-chain cytokines, especially interleukin (IL)-2 and IL-7, in influencing T cell homeostasis and proliferation, and discuss how immunotherapy with these cytokines may be beneficial to reconstitute the T cell compartment in the context of HIV-1 infection. The intriguing results of two large trials evaluating the efficacy of IL-2 in restoring immune function during HIV-1 infection are also discussed. In addition, we consider the promises and caveats of the first phase I/II clinical trials with IL-7 in HIV-1-infected patients and the knowledge that is still lacking in the field of T cell reconstitution through gamma-chain cytokines.

Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients.

BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.

53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

p53 binding protein-1 (53BP1) participates in checkpoint signaling during the DNA damage response (DDR) and during mitosis. In this study we report that 53BP1 aggregates in nuclear foci within syncytia elicited by the human immunodeficiency virus (HIV)-1 envelope. 53BP1 aggregation occurs as a consequence of nuclear fusion (karyogamy (KG)). It colocalizes partially with the promyelomonocytic leukemia protein (PML), and the ataxia telangiectasia mutated kinase (ATM), the two components of the DDR that mediate apoptosis induced by the HIV-1 envelope. ATM-dependent phosphorylation of 53BP1 on serines 25 and 1778 (53BP1S25P and 53BP1S1778P) occurs at these DNA damage foci. 53BP1S25P was also detected in syncytia present in the lymph nodes or frontal brain sections from HIV-1-infected carriers, as well as in peripheral blood mononucleated cells from HIV-1-infected individuals, correlating with viral load. Knockdown of 53BP1 caused HIV-1 envelope-induced syncytia to enter abnormal mitoses, leading to their selective destruction through mitochondrion-dependent and caspase-dependent pathways. In conclusion, depletion of 53BP1 triggers the demise of HIV-1-elicited syncytia through mitotic catastrophe.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

New insights on the role of apoptosis and autophagy in HIV pathogenesis.

Viruses manipulate host cells to ensure their own survival and, at late stages of the viral life cycle, they kill the infected target cell to ensure their propagation. In addition, some viruses induce a bystander killing, a viral strategy to escape from the host's innate and cognate defense systems. In HIV-infection, the disabling of the immune system is initially due to the preferential depletion by apoptosis of virus-specific CD4(+) T cells in lymphoid tissues, followed by the destruction of non-infected bystander cells. Both the extrinsic and the intrinsic pathways are activated, and this is the consequence of systemic immune activation. This review presents recent developments showing that the gastrointestinal tract is the major reservoir of infected cells and the site of rapid and profound loss of CD4 T cells, and that microbial translocation from the gastrointestinal tract is the cause of immune activation. Furthermore, apoptosis mechanisms involved in HIV-induced neuropathological disorders are discussed, including the role of syncytia that involve the sequential activation of ATM, p38MAPK and p53. Finally, HIV-associated dementia (HAD) was recently found in monkey models to be linked to inhibition of autophagy in neurons, suggesting that homeostasis of autophagy is a reliable security factor for neurons, and challenging the development of new therapeutics aimed at boosting neuronal autophagy to prevent HAD.

The tumor suppressor protein PML controls apoptosis induced by the HIV-1 envelope.

Promyelomonocytic leukemia (PML) is a prominent oncosuppressor whose inactivation is involved in the pathogenesis of hematological and epithelial cancers. Here, we report that PML aggregated in nuclear bodies in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. PML aggregation occurred after the fusion of nuclei (karyogamy) within syncytia but before the apoptotic program was activated. The aggregation of PML was detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Using a range of specific inhibitors of PML (the oncogenic PML/RARalpha fusion product or specific small interfering RNAs), we demonstrated that, in Env-elicited syncytia, PML was required for activating phosphorylation of ataxia telangiectasia mutated (ATM), which colocalized with PML in nuclear bodies, in a molecular complex that also involved topoisomerase IIbeta-binding protein 1. PML knockdown thus inhibited the ATM-dependent DNA damage response that culminates in the activation of p53, p53-dependent transcription of pro-apoptotic genes and cell death. Infection of CD4-expressing cells with HIV-1 also induced syncytial apoptosis, which could be suppressed by inhibiting PML. Altogether, these data indicate that PML activation is a critical early event that participates in the apoptotic demise of HIV-1-elicited syncytia.

Inhibition of naive Th1 CD4+ T cells by glatiramer acetate in multiple sclerosis.

We investigated whether glatiramer acetate (GA) treatment may affect Th1 differentiation at various T-cell maturation stages. Specifically, we analyzed the effect of in vivo GA treatment on intracellular synthesis of IL-2 and TNF-alpha by naive, memory and effector CD4(+) and CD8(+) T cells by five-colour flow cytometry. Our data indicate that GA treatment downregulates/normalizes an accelerated Th1 differentiation of CD4(+) T cells in RRMS patients at all stages of T-cell maturation. Most notably, we conclude that, by altering naive, unprimed CD4(+) T cells, GA treatment appears to affect T-cell differentiation, at least in part, in an antigen-independent manner.

Innate T-cell immunity in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes.

There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases. Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity. NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication. The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence. Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients. These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART). In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response. Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication. Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.

Longitudinal evolution of HIV-1-associated lipodystrophy is correlated to serum cortisol:DHEA ratio and IFN-alpha.

BACKGROUND: We have previously shown that lipid alterations in HIV-1-associated lipodystrophy (LD) are correlated with decreased serum dehydroepiandosterone (DHEA) and increased cortisol:DHEA ratio and IFN-alpha levels. OBJECTIVE: To evaluate in a longitudinal study whether steroid and cytokine modifications are associated with the evolution of physical changes and lipid alterations associated with LD. METHODS: Thirty-four HIV-1-positive men were followed during 32.5 +/- 4.0 months and tested at four time-points. The patients were subdivided into five groups according to physical changes and anthropometric measurements: LD-negative, initially LD-negative becoming LD-positive, LD-positive unchanged, aggravated or improved. Serum lipids, apolipoproteins, adrenal steroids and cytokines were measured and compared with baseline values. RESULTS: (1) LD aggravation is associated with persistent elevated lipids, a decrease in serum DHEA, an increase in cortisol:DHEA ratio and persistent high levels of IFN-alpha. (2) LD improvement is associated with normalization of serum lipids, an increase in serum DHEA leading to normalization in cortisol:DHEA ratio, and normalization of IFN-alpha levels. (3) In LD-positive men evolution of VLDL cholesterol is negatively correlated with DHEA (r = -0.56, P < 0.01) and positively with cortisol:DHEA ratio (r = 0.62, P < 0.004) and with IFN-alpha (r = 0.57, P < 0.01). (4) The switch to LD is associated with a decrease in serum DHEA. (5) Patients who remained LD-negative maintained normal lipids, elevated cortisol and DHEA, and normal cortisol:DHEA ratio and normal levels of IFN-alpha. CONCLUSIONS: This study indicates that cortisol:DHEA ratio and serum IFN-alpha levels are closely associated with clinical evolution and atherogenic lipid alterations in LD.

Innate T cell immunity to HIV-infection. Immunotherapy with phosphocarbohydrates, a novel strategy of immune intervention?

Natural T (NT) lymphocytes recognize infected cells or microbial compounds without the classical genetic restriction of polymorphic major histocompatibility complex (MHC) molecules. NT cells are mainly composed of alphabeta and gammadelta T lymphocytes that express natural killer (NK) receptors and recognize preferentially various nonpeptidic antigens. Similar to NK cells, NT lymphocytes can see and kill target cells deficient in the expression of one or more MHC class I molecules. NT cells expressing the alphabeta TCR can recognize lipid and lipoglycan antigens presented in the context of nonpolymorphic CD1 molecules, whereas phosphocarbohydrates and alkylamines induce constitutive response of Vgamma9Vdelta2 T cells. The stimulation of Vgamma9Vdelta2 T cells with phosphocarbohydrates induces the production of cytokines (IFNgamma and TNFalpha) and the release of chemokines with suppressive activity on HIV replication. In addition, stimulated Vgamma9Vdelta2 T cells exert a cytolytic activity against HIV-infected targets. In HIV-infected patients, a quantitative and qualitative alteration is observed early during the infection. Vgamma9Vdelta2 T cells are deleted and the remaining gammadelta cells are anergic. Th1 cytokines (IL-12 and IL-15) positively regulate cytokine production by Vgamma9Vdelta2 T cells but they are inefficient in restoring normal functions in patients' gammadelta T cells. Interestingly, partial restoration of the immune system under highly active antiretroviral therapies (HAART) is associated to the recovery of functional Vgamma9Vdelta2 T cells. A large panel of phosphocarbohydrates able to selectively stimulate Vgamma9Vdelta2 T cells is currently available, and preliminary experiments in monkeys suggest their in vivo efficacy in helping to control SIV replication. These observations prompt the question of new immune intervention involving molecules that stimulate NT cells.

Increased serum interferon alpha in HIV-1 associated lipodystrophy syndrome.

BACKGROUND: A syndrome of lipodystrophy (LD) associated with peripheral lipoatrophy and central/visceral adiposity has been reported in HIV-1-infected patients treated by combined antiretroviral therapy (ART). Lipid metabolism is partly regulated by both steroid hormones and cytokines and we have previously reported that dyslipidaemia in LD-positive men is correlated to an increase in cortisol : DHEA ratio (Christeff et al., AIDS 1999;13:2251). In this study we questioned whether it is also related to cytokine perturbations. MATERIALS AND METHODS: A cross-sectional study was performed on 42 HIV-1-positive men on ART, 27 of whom had symptoms of LD, defined by computed tomography scan. Serum cytokines (IFN-alpha, TNF-alpha, sTNF-RI, sTNF-RII, IL-6, IL-1beta and IL-2) and lipids [cholesterol, triglycerides (TG) and their subclasses], and apolipoproteins (Apo), were determined. RESULTS: Serum IFN-alpha was markedly increased in LD-positive compared with LD-negative men and controls. IL-6 and TNF-alpha concentrations were also significantly elevated in HIV-positive men compared to controls but the levels of these cytokines did not differ between the two groups of patients. A significant positive correlation was found between accumulation of IFN-alpha and increased levels of cholesterol, TG, VLDL cholesterol, VLDL TG, ApoB and ApoB-ApoA1 ratio. A multivariate forward-performed analysis revealed that IFN-alpha is the best marker for lipid perturbations associated to LD, followed by insulin and cortisol : DHEA ratio. CONCLUSIONS: This study demonstrates an association between serum IFN-alpha and lipid alterations in LD-positive men. The concomittant action of IFN-alpha and cortisol : DHEA ratio is probably one of the mechanisms responsible for hyperlipidaemia in LD syndrome.