CD4 T Follicular Helper Cells and HIV Infection: Friends or Enemies?

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection.JE from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS) and from The Canadian HIV Cure Enterprise Team Grant HIG-13305 from the Canadian Institutes of Health Research (CIHR) in partnership with CANFAR and IAS. FM is supported by a fellowship from Fondation du CHU de Québec. CB and YF are supported by fellowships from ANRS. JE acknowledges the support of the Canada Research Chair program. RS is supported by FCT—Fundaçao para a Ciência e a Tecnologia/MEC—Ministério da Educaçao e Ciência através de fundos nacionais e quando aplicavel cofinanciado pelo FEDER, no âmbito do Acordo de Parceria PT2020 referente à unidade de investigaçao n°4293. RS is supported by the Fundaçao para a Ciência e a Tecnologia (FCT) (IF/00021/2014)info:eu-repo/semantics/publishedVersio

Stepwise Release of Biologically Active HMGB1 during HSV-2 Infection

BACKGROUND: High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data. CONCLUSIONS: These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection

A High-Sensitivity Method for Detection and Measurement of HMGB1 Protein Concentration by High-Affinity Binding to DNA Hemicatenanes

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies

Epstein–Barr Virus BALF0 and BALF1 Modulate Autophagy

International audienceAutophagy is an essential catabolic process that degrades cytoplasmic components within the lysosome, therefore ensuring cell survival and homeostasis. A growing number of viruses, including members of the Herpesviridae family, have been shown to manipulate autophagy to facilitate their persistence or optimize their replication. Previous works showed that the Epstein-Barr virus (EBV), a human transforming gammaherpesvirus, hijacked autophagy during the lytic phase of its cycle, possibly to favor the formation of viral particles. However, the viral proteins that are responsible for an EBV-mediated subversion of the autophagy pathways remain to be characterized. Here we provide the first evidence that the BALF0/1 open reading frame encodes for two conserved proteins of the Bcl-2 family, BALF0 and BALF1, that are expressed during the early phase of the lytic cycle and can modulate autophagy. A putative LC3-interacting region (LIR) has been identified that is required both for BALF1 colocalization with autophagosomes and for its ability to stimulate autophagy

Dipyridamole as a new drug to prevent Epstein-Barr virus reactivation

International audienceEpstein-Barr virus (EBV) is a widely distributed gamma-herpesvirus that has been associated with various cancers mainly from lymphocytic and epithelial origin. Although EBV-mediated oncogenesis has been associated with viral oncogenes expressed during latency, a growing set of evidence suggested that antiviral treatments directed against EBV lytic phase may contribute to prevent some forms of cancers, including EBV-positive Post-Transplant Lymphoproliferative Diseases. It is shown here that dipyridamole (DIP), a safe drug with favorable and broad pharmacological properties, inhibits EBV reactivation from B-cell lines. DIP repressed immediate early and early genes expression mostly through its ability to inhibit nucleoside uptake. Considering its wide clinical use, DIP repurposing could shortly be evaluated, alone or in combination with other antivirals, to treat EBV-related diseases where lytic replication plays a deleterious role

Detection of HMGB1 in human serum.

<p>(A) On the left half of the gel, complexes obtained with increasing amounts of pure HMGB1: 0 pg, 10 pg, 100 pg, 1 ng, 10 ng (competitor DNA: 2 µg double strand plus 1 µg single strand per sample). The next five samples contain the same amounts of HMGB1 plus an anti-HMGB1 antibody, showing the supershift of the specific complexes (since pure HMGB1 was used here, and as antibodies were polyclonal and present in excess, the partial supershifting is most likely due to a low or moderate affinity of part of the antibodies). On the right-hand side of the gel, the same experiment was performed with the same amounts of HMGB1 and competitor DNA mixed with 1 µL of serum from a healthy individual. The overall pattern of shifted and supershifted bands is identical, over a background that contains a number of non-specific extra bands, despite the use of a large amount of nonspecific competitor DNA. (B) Purification of HMGB1 by PCA treatment. The left-hand part of the gel shows the interactions of hcDNA with the indicated amounts of pure HMGB1, from 1 pg to 100 pg. On the right-hand part of the gel, samples containing the same concentrations of HMGB1, plus 50 mg/mL BSA to mimic an HMGB1-free serum, were treated with PCA and neutralized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002855#s2" target="_blank">Materials and Methods</a>, then assayed for hcDNA binding. Note that the hcDNA-binding activity is recovered without any loss after the PCA treatment procedure.</p

HMGB1 assay in serums from patients.

<p>(A) without PCA treatment, or (B) after sample purification by PCA treatment. On each gel the five samples on the left, prepared with known amounts of pure HMGB1, were used to calibrate the method. The 15 serums tested are labeled a-o, serum a originating from a healthy individual, serums b-o from randomly selected patients in intensive care unit (d,g,h,i,k,l,n: sepsis; b,c,e,f,j,m,o: septic shock). Note the lower sensitivity, higher background, and non-specific bands (arrowheads) obtained with crude serums, as compared with the strong increase in sensitivity and marked background decrease after treatment of serums with PCA. (C) Comparison of band-shift assay with ELISA. Concentrations measured by ELISA are plotted against band-shift assay data obtained after PCA treatment of serums, on a double logarithmic scale. Each circled letter on the diagram represents the corresponding serum as assayed in (B).</p

HMGB1 protein assay in culture medium of epithelial human cells infected by herpes simplex virus type 2.

<p>(A) Cell culture medium assayed at the indicated times after infection. At days 3 and 4, HMGB1 concentration was so high that the hcDNA probe was saturated, requiring a tenfold dilution of the sample for determination of HMGB1 concentrations (right panel). (B) Gel and calibration curve, obtained with known amounts of pure HMGB1 under identical conditions.</p