Establishment of Murine Gammaherpesvirus latency in B cells is not a stochastic event

Copyright: © 2014 Decalf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Murid γ-herpesvirus-4 (MuHV-4) promotes polyclonal B cell activation and establishes latency in memory B cells via unclear mechanisms. We aimed at exploring whether B cell receptor specificity plays a role in B cell susceptibility to viral latency and how this is related to B cell activation. We first observed that MuHV-4-specific B cells represent a minority of the latent population, and to better understand the influence of the virus on non-MuHV-4 specific B cells we used the SWHEL mouse model, which produce hen egg lysozyme (HEL)-specific B cells. By tracking HEL+ and HEL- B cells, we showed that in vivo latency was restricted to HEL- B cells while the two populations were equally sensitive to the virus in vitro. Moreover, MuHV-4 induced two waves of B cell activation. While the first wave was characterized by a general B cell activation, as shown by HEL+ and HEL- B cells expansion and upregulation of CD69 expression, the second wave was restricted to the HEL- population, which acquired germinal center (GC) and plasma cell phenotypes. Antigenic stimulation of HEL+ B cells led to the development of HEL+ GC B cells where latent infection remained undetectable, indicating that MuHV-4 does not benefit from acute B cell responses to establish latency in non-virus specific B cells but relies on other mechanisms of the humoral response. These data support a model in which the establishment of latency in B cells by γ-herpesviruses is not stochastic in terms of BCR specificity and is tightly linked to the formation of GCs.Research in JPS's lab was supported by a grant from the Portuguese Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-MII/099314/2008). JD was recipient of a FCT fellowship.info:eu-repo/semantics/publishedVersio

Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients

Plasmacytoid dendritic cells (pDCs) are the professional type I interferon (IFN)-producing cells, and upon activation they traffic to lymph organs, where they bridge innate and adaptive immunity. Using multianalyte profiling (MAP), we have mapped the key chemokines and cytokines produced in response to pDC activation, taking into consideration the role of autocrine IFN, as well as paracrine effects on other innate cells (e.g., monocytes and conventional DCs). Interestingly, we identify four distinct cytokine/chemokine loops initiated by Toll-like receptor engagement. Finally, we applied this analytic approach to the study of pDC activity in chronic hepatitis C patients. Based on the activation state of pDCs in fresh blood, the lack of agonistic activity of infectious virions, the production of a broad array of cytokines/chemokines once stimulated, and the direct effects of pDCs on other PBMCs, we conclude that the pDCs from hepatitis C virus (HCV)-infected individuals are fully functional and are, indeed, a viable drug target. In sum, this study provides insight into the use of MAP technology for characterizing cytokine networks, and highlights how a rare cell type integrates the activation of other inflammatory cells. Furthermore, this work will help evaluate the therapeutic application of pDC agonists in diseases such as chronic HCV infection

Defining plasmacytoid dendritic cells biology (from fundamental immunology to basics for chronic hepatitis C immunotherapy)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Plasmacytoid dendritic cells move down on the list of suspects: In search of the immune pathogenesis of chronic hepatitis C

International audienceChronic hepatitis C is a major public health problem. Despite numerous clinical studies in humans and experimental observations made in chimpanzees, hepatitis C pathogenesis remains poorly understood. Here, we review the clinical features of acute and chronic disease, and discuss the role of the immune system in the pathogenesis of disease. Many are aware of the dual role of T cells: responsibility for clearance of the virus during acute phase; and liver injury during chronic phase. Nonetheless, there is an emerging belief that failure to prime HCV-specific T cells is responsible for the failure to spontaneously clear the virus, and possibly, for the lack of response to pegylated-IFNalpha(2a)/ribavirin therapy. We have focused on the latest suspects, plasmacytoid dendritic cells (pDCs), considered to be the professional type I IFNs producing cells. We review the somewhat contradictory data regarding the functional capacity of pDCs in chronic HCV patients and argue that, while lower in relative concentration as compared to healthy individuals, they are not defective in their ability to initiate an innate inflammatory response. Thus, instead of being the culprit, pDCs may in fact represent a novel therapeutic target in order to improve upon existing therapies for treating HCV patients

Dynamic Changes of Post-Translationally Modified Forms of CXCL10 and Soluble DPP4 in HCV Subjects Receiving Interferon-Free Therapy.

Serum levels of the interferon (IFN)-stimulated chemokine CXCL10 are increased during chronic HCV infection and associate with outcome of IFN-based therapy. Elevated levels of NH2-terminal truncated CXCL10 (3-77aa), produced by DPP4 cleavage, negatively associate with spontaneous clearance of acute HCV infection and sustained virological response (SVR) with IFN-based therapy for chronic infection. The association of different CXCL10 forms and DPP4 with outcome during IFN-free HCV therapy has not been examined. Using novel Simoa assays, plasma was analyzed from HCV genotype-1 (GT1) subjects who relapsed (n = 11) or achieved SVR (n = 10) after sofosbuvir and ribavirin (SOF/RBV) treatment, and from SOF/RBV relapsers who achieved SVR with a subsequent SOF/ledipasvir regimen (n = 9). While the NH2-truncated form of CXCL10 was elevated in HCV infection relative to healthy controls, pre-treatment plasma concentrations of CXCL10 forms failed to stratify subjects based on treatment outcome to IFN-free regimens. However, a trend (statistically non-significant) towards elevated higher levels of total and long CXCL10 was observed pre-treatment in subjects who relapsed. All forms of CXCL10 decreased rapidly following treatment initiation and were again elevated in subjects who experienced HCV relapse, indicating that CXCL10 production may be associated with active viral replication. While soluble DPP4 (sDPP4) and NH2-truncated CXCL10 concentrations were highly correlated, on-treatment sDPP4 levels and activity declined more slowly than CXCL10, suggesting differential regulation. These data suggest post-translationally modified forms of CXCL10 will not support the prediction of treatment outcome in HCV GT1 subjects treated with SOF/RBV

SW_HEL mice mount a long-lasting anti-MuHV-4 antibody response.

<p>SW_HEL mice were inoculated intranasally with PBS (n = 2) or MuHV-4 (n = 6). At indicated time points, mice were bled and sera were analyzed by ELISA to monitor levels of circulating anti-MuHV-4 and anti-HEL IgG₁ (red triangles), IgG_2a (blue squares) and IgG_2b (green circles). For MuHV-4 infected mice, 3 independent experiments with 2 mice in each were analyzed. As no standard were available to quantify these antibodies, samples of different time points from identical mice were run together to be able to compare the absorbance at 405 nm.</p

Immunosuppressive Signature of Cutaneous Mycobacterium ulcerans Infection in the Peripheral Blood of Patients with Buruli Ulcer Disease

International audienceBuruli ulcer disease (BUD) is an emerging human disease caused by infection with Mycobacterium ulcerans, which leads to the development of necrotic skin lesions. The pathogenesis of the ulcer is closely associated with the production of mycolactone, a diffusible cytotoxin with immunomodulatory properties. To identify immunological correlates of BUD, we performed a broad screen of inflammatory mediators in serum samples and stimulated whole-blood supernatants of patients. We found that patients with active ulcers displayed a distinctive profile of immune suppression, marked by the down-modulation of selected chemokines and an impaired capacity to produce Th1, Th2, and Th17 cytokines on stimulation with mitogenic agents. These immunological defects were induced early in the disease and resolved after anti-BUD therapy, establishing their association with the presence of M. ulcerans. Interestingly, some of the defects in cytokine and chemokine response could be mimicked in vitro by incubation of CD4(+) peripheral blood lymphocytes with mycolactone. Our findings support the hypothesis that mycolactone contributes to bacterial persistence in human hosts by limiting the generation of adaptive cellular responses. Moreover, we identified immunological markers of BUD, which may be helpful for confirmatory diagnosis of the disease and, especially, for monitoring the response to antibiotic treatment

MuHV-4 latent infection is restricted to polyclonal HEL⁻ B cells.

<p>SW_HEL mice were left untreated or infected for 14 days with YFP-MuHV-4 virus at 10⁴ PFU. Splenocytes were isolated and analyzed by FACS or sorted for plaque assay and limiting dilution PCR analysis. HEL⁺ and HEL⁻ B cells were identified based on HEL-A647 binding and CD19 expression. (A) On top are representative FACS plots showing the percentage of YFP⁺ cells in both populations at 0 and 14 dpi. Data are compiled in the graphics below showing percentage of YFP⁺ cells in HEL⁺ (grey circles) and HEL⁻ (white circles) B cells at day 0 (n = 12) and 14 (n = 16) dpi. The bar represents the average percentage. (B) To detect presence of latent MuHV-4, a plaque assay was performed on sorted HEL⁺ and HEL⁻ B cells 14 dpi (n = 8). # indicates that no plaques were detected in HEL⁺ B cells and based on the average number of HEL+ B cells plated, we estimate the level of latent infection to be <1 PFU/382000 in this population. The analysis of lysed cells showed an absence of preformed viral particles in both populations. (C) Limiting dilution PCR was performed to detect viral genome in sorted HEL⁺ and HEL⁻ B cells from day 14 infected SW_HEL mice. Data are representative of two independent experiments in which splenocytes were pooled before sorting (n = 3 & n = 2). The frequency of cells harboring MuHV-4 DNA is shown, # indicates signal below the assay's limit of detection. We evaluated the frequency of MuHV-4 DNA⁺ cells in HEL⁺ B cells to be ≤1/36770 and ≤1/102891 for the two experiments we performed. Error bars indicate the 95% confidence interval. Purity of sorted cells was systematically above 90%.</p

HEL⁺ and HEL⁻ B cells are both infected by MuHV-4 in vitro.

<p>In vitro infection by co-culture assay was performed as described in the material and methods. Briefly, freshly isolated SW_HEL splenocytes were exposed to free viruses or co-culture with BHK-21 or RAW-264 cells previously infected with EF1α-eGFP⁺ or EF1α-eGFP⁺-gp150⁻ MuHV-4. GFP expression in HEL⁺ and HEL⁻ B cells was monitored by FACS after 48 h of co-culture. Representative FACS plots are shown on top, compiled data representing average percentage and standard deviation are shown below. Splenocytes were identified based on FSC SSC parameters, excluding BHK-21 and RAW-264. Cells were then gated on CD19⁺ CD11b⁻ and GFP expression was monitored in HEL⁺ and HEL⁻ B cell. Data were obtained from two independent experiments with two splenocytes suspensions in each.</p

Proliferation and transient activation of total B cells.

<p>Spleens and CLN from YFP-MuHV-4 infected SW_HEL mice were harvested at 0, 7, 14 and 21 dpi and cells were stained with CD69 PE, CD19 APC-Cy7 and HEL-A647. (A) Live cells were enumerated, and the number of HEL⁺ and HEL⁻ B cells was established based on their frequency obtained from the FACS data. (B) Representative FACS histograms showing CD69 expression on HEL⁺ (grey curves) and HEL⁻ (white curves) B cells isolated from CLN and spleen at the different time points are shown. CD69 was monitored by measuring the Geo MFI and compiled values are shown in the graphic below. (C) On the same samples, the frequency of CD69^high cells was evaluated. Representative FACS plots are shown and compiled percentages are presented in the graphic below. These data were obtained from two independent experiments, with a total 5 to 6 mice per time point. In the graphics, mean values are reported and error bars represent the standard deviation.</p