Research Group
- Dr Franck Halary, Principal Investigator
- Mr Jérémy Reignier, Co-Investigator
- Dr Iulia Mocan, Co-Investigator
- Prof. Jean-François Moreau, Research Associate
- Prof. Pierre Merville, Research Associate
Location
- Université de Nantes, Nantes, France
Title
- Study of CMV/DC Interplays in Human Renal Allograft Transplantation
Immunosuppressive drugs have been extensively used for several decades in transplantation to decrease acute graft rejection. By weakening cell-mediated immune responses in recipients, opportunistic pathogens such as cytomegalovirus (CMV) have arisen and directly promoted transplant failure and rejection. To avoid CMV infection, anti-viral drugs are usually given to newly transplanted patients in addition to immunosuppressive medication. However, prophylactic therapies do not definitively clear the virus since anti-viral drugs only target replicating virus. Of note, we have recently reported that dendritic cells (DCs) were able to capture CMV particles. These internalized virions not only escaped drugs but also kept their infectivity for an extended time. This previous work suggested that in vivo DCs could likely capture CMV via specific lectins such as DC-SIGN at inoculation or reactivation sites and protect it from neutralization by a rapid internalization into yet uncharacterized endocytic vesicles.
Our present research project has been designed and granted by the ROTRF to study 1) the cell fate of DC-internalized CMV and how the virus escapes from a rapid immune inactivation, 2) how immunosuppressors, with or without antiviral drugs, may modify the DC phenotype and by the way modify the contribution of DCs to an enhanced or decreased CMV disease.
Regarding the first point, during the last eight months, we performed experiments to delineate the DC-SIGN-mediated CMV internalization into DCs. We used drugs known to interfere with endocytosis or cytoskeleton movement on the infection rate of monocyte-derived DCs (moDCs). Briefly, infected moDCs were immobilized on glass slides before being stained for CMV antigens. Infected versus total cells were counted on digitalized images and allowed us to determine the mean infection rate for each experimental conditions. Based on this, we proposed that CMV particles were internalized into DC through macropinocytosis (Fig. 1). Consistent with our previous work, we also showed by confocal and electron microscopy that engulfed virions were either stored in partially identified vesicles for at least several hours without losing their infectious abilities or conveyed to the nucleus where they could initiate a productive infection.
The second issue is related to the study of phenotypic and functional (especially CMV infection susceptibility) changes that could occur in DCs following their exposure to immunosuppressive and anti-viral drugs in vitro. We obtained preliminary results indicating that several commonly used drugs such as corticoids, azathioprine and mycophenolate might hamper DC differentiation. Treated DCs seemed to harbour an intermediate phenotype as documented by flow cytometry. Significant differences have been mainly observed for morphological parameters (SSC and FSC) as well as for DC-SIGN, HLA class II, B7.1/2 and CD83 mean fluorescent intensities. Other experiments have been undertaken to evaluate how these modifications could influence the CMV infection rate of moDCs by using recombinant CMV strains. To go further, we intend to compare our in vitro data (phenotypic changes) with clinical data of the DIVAT (Données Informatique VAlidées en Transplantation) database.
Figure 1. Schematic hypothetical model of the macropinocytosis-driven internalization of CMV into human monocyte-derived DC. All steps, from the capture to the capsid release into the cytoplasm and the initiation of the viral lytic cycle (from the top to bottom and from left to right), are displayed in boxes with capital letters. Some of the inhibitors we used are shown in small red characters.
