Dynamic LTR retrotransposon transcriptome landscape in septic shock patients.

BACKGROUND: Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Numerous studies have explored the complex and dynamic transcriptome modulations observed in sepsis patients, but a large fraction of the transcriptome remains unexplored. This fraction could provide information to better understand sepsis pathophysiology. Multiple levels of interaction between human endogenous retroviruses (HERV) and the immune response have led us to hypothesize that sepsis is associated with HERV transcription and that HERVs may contribute to a signature among septic patients allowing stratification and personalized management. METHODS: We used a high-density microarray and RT-qPCR to evaluate the HERV and Mammalian Apparent Long Terminal Repeat retrotransposons (MaLR) transcriptome in a pilot study that included 20 selected septic shock patients, stratified on mHLA-DR expression, with samples collected on day 1 and day 3 after inclusion. We validated the results in an unselected, independent cohort that included 100 septic shock patients on day 3 after inclusion. We compared septic shock patients, according to their immune status, to describe the transcriptional HERV/MaLR and conventional gene expression. For differential expression analyses, moderated t tests were performed and Wilcoxon signed-rank tests were used to analyze RT-qPCR results. RESULTS: We showed that 6.9% of the HERV/MaLR repertoire was transcribed in the whole blood, and septic shock was associated with an early modulation of a few thousand of these loci, in comparison to healthy volunteers. We provided evidence that a subset of HERV/MaLR and conventional genes were differentially expressed in septic shock patients, according to their immune status, using monocyte HLA-DR (mHLA-DR) expression as a proxy. A group of 193 differentially expressed HERV/MaLR probesets, tested in an independent septic shock cohort, identified two groups of patients with different immune status and severity features. CONCLUSION: We demonstrated that a large, unexplored part of our genome, which codes for HERV/MaLR, may be linked to the host immune response. The identified set of HERV/MaLR probesets should be evaluated on a large scale to assess the relevance of these loci in the stratification of septic shock patients. This may help to address the heterogeneity of these patients.

In Vitro Characterization and In Vivo Effectiveness of Ebola Virus Specific Equine Polyclonal F(ab')2.

There is no vaccine or approved therapy against lethal Ebola virus (EBOV). We investigated a proven technology platform to produce polyclonal IgG fragments, F(ab')2, against EBOV. Horses immunized with nanoparticles harboring surface glycoprotein trimers of EBOV-Zaire/Makona produced anti-Ebola IgG polyclonal antibodies with high neutralization activity. Highly purified equine anti-Ebola F(ab')2 showed strong cross-neutralization of 2 Zaire EBOV strains (Gabon 2001 and Makona) and in vivo 3 or 5 daily F(ab')2 intraperitoneal injections provided 100% protection to BALB/c mice against lethal EBOV challenge. Rapid preparation of purified equine anti-Ebola F(ab')2 offers a potentially efficient therapeutic approach against EBOV disease in humans.

Chikungunya virus mobilizes the apoptotic machinery to invade host cell defenses.

Chikungunya virus (CHIKV) surprised medical workers by a massive outbreak in the Indian Ocean region, reaching Europe in 2007, with exceptional pathologies in infants and elderly patients. Although CHIKV was recently shown to persist in myoblasts, monocytes, and macrophages, we argued that robust antiviral mechanisms, including apoptosis, are essential to ward off the virus. Herein, we tested the capacity of CHIKV to mobilize the apoptotic machinery in HeLa cells as well as primary fibroblasts, making use of several inhibitors of caspases, cell blebbing, and engulfment of the apoptotic blebs by neighboring cells. CHIKV triggered apoptosis through intrinsic and extrinsic pathways. Bystander apoptosis was also evidenced in neighboring cells in a caspase-8-dependent manner. Remarkably, by hiding in apoptotic blebs, CHIKV was able to infect neighboring cells. In HeLa cells, these events were inhibited specifically by zVAD-fmk and DEVD-cho (caspase inhibitors), blebbistatin, Y-27632 (ROCK inhibitor), and genistein, annexin V, and cytochalasin B (inhibitors of blebbing and engulfment). These CHIKV-apoptotic blebs were also capable of infecting macrophages (primary cultures, MM6- and THP1-PMA differentiated cells) otherwise refractory to infection by CHIKV alone. Remarkably, viral replication in macrophages did not yield a proinflammatory response. We describe a novel infectious mechanism by which CHIKV invades host cells and escapes the host response.

Deciphering the differential response of two human fibroblast cell lines following Chikungunya virus infection.

BACKGROUND: Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN) response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I) to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs). We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response. METHODS: Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV. RESULTS: Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI). We found that the expression of antiviral genes (RIG-I, IFN-ß, ISG54 and ISG56) was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines. CONCLUSION: Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by fibroblasts to control CHIKV infection, replication and spreading from cell to cell.

Persistent chronic inflammation and infection by Chikungunya arthritogenic alphavirus in spite of a robust host immune response.

Alphaviruses, including Chikungunya virus (CHIKV), produce a transient illness in humans, but severe forms leading to chronic incapacitating arthralgia/arthritis have been reported by mechanisms largely ill-characterized. The pathogenesis of CHIKV was addressed in a prospective cohort study of 49 hospitalized patients from Reunion Island subsequently categorized into two distinct groups at 12 mo postinfection. Comprehensive analyses of the clinical and immunological parameters throughout the disease course were analyzed in either the "recovered" or the "chronic" groups to identify prognostic markers of arthritis-like pathology after CHIKV disease. We found that the chronic group consisted mainly of more elderly patients (>60 y) and with much higher viral loads (up to 10(10) viruses per milliliter of blood) during the acute phase. Remarkably, a rapid innate immune antiviral response was demonstrated by robust dendritic/NK/CD4/CD8 cell activation and accompanied by a rather weak Th1/Th2 cytokine response in both groups. Interestingly, the antiviral immune response witnessed by high levels of IFN-alpha mRNA in PBMCs and circulating IL-12 persisted for months only in the chronic group. CHIKV (RNA and proteins) was found in perivascular synovial macrophages in one chronic patient 18 mo postinfection surrounded by infiltrating NK and T cells (CD4(++) but rare cytotoxic CD8). Fibroblast hyperplasia, strong angiogenesis, tissue lesions given the high levels of matrix metalloproteinase 2, and acute cell death [high cleaved poly(ADP-ribose) polymerase staining] were observed in the injured synovial tissue. These observed cellular and molecular events may contribute to chronic arthralgia/arthritis targeted by methotrexate used empirically for effective treatment but with immunosuppressive function in a context of viral persistence.

Chikungunya triggers an autophagic process which promotes viral replication.

BACKGROUND: Chikungunya Virus (ChikV) surprised by a massive re-emerging outbreak in Indian Ocean in 2006, reaching Europe in 2007 and exhibited exceptional severe physiopathology in infants and elderly patients. In this context, it is important to analyze the innate immune host responses triggered against ChikV. Autophagy has been shown to be an important component of the innate immune response and is involved in host defense elimination of different pathogens. However, the autophagic process was recently observed to be hijacked by virus for their own replication. Here we provide the first evidence that hallmarks of autophagy are specifically found in HEK.293 infected cells and are involved in ChikV replication. METHODS: To test the capacity of ChikV to mobilize the autophagic machinery, we performed fluorescence microscopy experiments on HEK.GFP.LC3 stable cells, and followed the LC3 distribution during the time course of ChikV infection. To confirm this, we performed electron microscopy on HEK.293 infected cells. To test the effect of ChikV-induced-autophagy on viral replication, we blocked the autophagic process, either by pharmacological (3-MA) or genetic inhibition (siRNA against the transcript of Beclin 1, an autophagic protein), and analyzed the percentage of infected cells and the viral RNA load released in the supernatant. Moreover, the effect of induction of autophagy by Rapamycin on viral replication was tested. RESULTS: The increasing number of GFP-LC3 positive cells with a punctate staining together with the enhanced number of GFP-LC3 dots per cell showed that ChikV triggered an autophagic process in HEK.293 infected cells. Those results were confirmed by electron microscopy analysis since numerous membrane-bound vacuoles characteristic of autophagosomes were observed in infected cells. Moreover, we found that inhibition of autophagy, either by biochemical reagent and RNA interference, dramatically decreases ChikV replication. CONCLUSIONS: Taken together, our results suggest that autophagy may play a promoting role in ChikV replication. Investigating in details the relationship between autophagy and viral replication will greatly improve our knowledge of the pathogenesis of ChikV and provide insight for the design of candidate antiviral therapeutics.

Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4.

HIV-1 envelope glycoproteins (Env), expressed at the cell surface, induce apoptosis of uninfected CD4+ T cells, contributing to the development of AIDS. Here we demonstrate that, independently of HIV replication, transfected or HIV-infected cells that express Env induced autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. The same phenomena occurred in a T cell line and in transfected HEK.293 cells that expressed both wild-type CXCR4 and a truncated form of CD4 that is unable to bind the lymphocyte-specific protein kinase Lck. Env-mediated autophagy is required to trigger CD4+ T cell apoptosis since blockade of autophagy at different steps, by either drugs (3-methyladenine and bafilomycin A1) or siRNAs specific for Beclin 1/Atg6 and Atg7 genes, totally inhibited the apoptotic process. Furthermore, CD4+ T cells still underwent Env-mediated cell death with autophagic features when apoptosis was inhibited. These results suggest that HIV-infected cells can induce autophagy in bystander CD4+ T lymphocytes through contact of Env with CXCR4, leading to apoptotic cell death, a mechanism most likely contributing to immunodeficiency.

Efficacy of a specific polyclonal equine F(ab')2 against avian influenza (H5N1) in ferrets: synergy with oseltamivir.

AIM: Current therapies against avian influenza (H5N1) provide limited clinical benefit. FBF-001 is a highly purified equine polyclonal immunoglobulin fragment against H5N1. METHODS: Using a ferret model of severe acute H5N1 infection, we assessed FBF-001 when administered on the same day or 1 day after viral challenge, in comparison with oseltamivir therapy. RESULTS: Untreated animals died 2-3 days after challenge. FBF-001 prevented most severe illness and reduced nasal viral load, with best efficacy when administered on the day of viral challenge. Oseltamivir and FBF-001 had synergistic impact on survival. CONCLUSION: FBF-001 prevented severe consequences of lethal H5N1 challenge in ferrets by controlling viral replication, an effect synergistic to oseltamivir. FBF-001 has recently been granted EMA orphan drug status.

Mitochondria-dependent apoptosis in T-cell homeostasis.

T-cell homeostasis is maintained by balancing the proliferation and destruction of lymphocytes at multiple steps during the life of an individual. Regulated mitochondria-dependent apoptosis is essential for both the development and the subsequent maintenance of the immune system, in that it keeps the total number of lymphocytes constant. Firstly, during thymic development, sequential stages of T-cell maturation require strict control of T-cell selection, and secondly, apoptosis is essential in controlling the massive expansion of antigen-specific T-cells after their activation. Failure in each of these steps can lead to pathologies, while drugs that target apoptosis could have therapeutic benefit.

[Contributions and limits of the diagnosis of human African trypanosomiasis]. / Apports et limites du diagnostic de la trypanosomiase humaine africaine.

Human African trypanosomiasis, or sleeping sickness, is still a worrying problem in Africa. Sleeping sickness is a disease for which a systematic monitoring is necessary, particularly for the trypanosomiasis caused by Trypanosoma brucei gambiense, which is characterized by a long asymptomatic stage. In the absence of specific clinical signs, mass screening of populations remains the only way to control the disease and to avoid its spreading. The lack of sensitivity and specificity of the diagnosis tests classically used led to the development of molecular tools. PCR amplification of parasite specific sequences has considerably improved the diagnostic of the parasitic infection, the stage diagnosis as well as the post-therapeutic follow-up. But there are limits with a use in routine and research is still necessary to make PCR a real tool for control of sleeping sickness.

Chikungunya fever: CNS infection and pathologies of a re-emerging arbovirus.

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes an acute symptomatic illness with fever, skin rash, and incapacitating arthralgia, which can evolve into chronic rheumatoid arthritis in elderly patients. This is a tropical disease originally described in central/east Africa in the 1960s, but its 2004 re-emergence in Africa and rapid spread in lands in and around the Indian Ocean (Reunion island, India, Malaysia) as well as Europe (Italy) led to almost 6 million cases worldwide. The risk of importation and spreading diseases with long-term sequelae is even greater today given the global distribution of the vectors (including in the Americas), increased tourism and the apparent capacity of CHIKV to produce high levels of viremia (10(9)-10(12) virus/ml of blood) and new mutants. CHIKV-associated neuropathology was described early in the 1960s, but it is the unprecedented incidence rate in Indian Ocean areas with efficient clinical facilities that allowed a better description of cases with severe encephalitis, meningoencephalitis, peripheral neuropathies and deaths among newborns (mother-to-child infection), infants and elderly patients. Death rates following CHIKV infection were estimated at 1:1000 cases in la Reunion's outbreak. These clinical observations have been corroborated by experimental infection in several mouse models, leading to CNS pathologies. We further describe in this review the capacity of CHIKV to infect neurons and glial cells, delineate the fundamental innate (intrinsic) immune defence mechanisms to protect from infection and argue about the possible mechanisms involved in the encephalopathy.

Chikungunya virus takes centre stage in virally induced arthritis: possible cellular and molecular mechanisms to pathogenesis.

Chikungunya virus (CHIKV) causes an acute symptomatic illness with fever, skin rash (hypersensitivity vasculitis), incapacitating arthralgia which can evolve to chronic arthritis in elderly patients. Clinical observations from cohort studies have been corroborated with data from experimental infection in several mouse and non-human primate models as discussed herein.

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells.

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.

Autophagy and CD4+ T lymphocyte destruction by HIV-1.

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.

Identification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation.

The chemokine SDF-1alpha transduces G(i)-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a G(i)-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1alpha binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr(135) in the DRY motif at the N terminus of ICL2 nor the Tyr(65) and Tyr(76) in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1alpha binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.