Epstein-Barr Virus Induces Activation of Infammatory Markers Via the TLR8 Transduction Pathway in Infected Scleroderma Monocytes

Antonella Farina¹, Giovanna Peruzzi^2,3, Valentina Lacconi⁴, Edoardo Rosato⁵, Silvia Quarta⁶, Stefania Morrone¹, Alberto Faggioni¹, Maria Trojanowska⁴ and Giuseppina Alessandra Farina⁴, ¹Department of Experimental Medicine, Sapienza University, Department of Experimental Medicine, Rome, Italy, ²Istituto Italiano di Tecnologia (IIT), CLNS@Sapienza, Rome, Italy, ³Istituto Italiano di Tecnologia (IIT), CLNS@Sapienza, Viale Regina Elena 291, 00161, Rome, Italy, Rome, Italy, ⁴Arthritis Center, Boston University, Arthritis Center, Boston, MA, ⁵Department of Clinical Medicine, Clinical Immunology Unit,, Sapienza University,Department of Clinical Medicine, Rome, Italy, ⁶Department of Clinical Medicine, Clinical Immunology Unit., Sapienza University,Department of Clinical Medicine, Rome, Italy

Meeting: 2015 ACR/ARHP Annual Meeting

Date of first publication: September 29, 2015

Keywords: Inflammation, monocytes, Systemic sclerosis, toll-like receptors and viruses

Session Information

Date: Tuesday, November 10, 2015

Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics II

Session Type: ACR Concurrent Abstract Session

Session Time: 2:30PM-4:00PM

Background/Purpose:

Monocytes from patients with systemic sclerosis (Scleroderma, SSc), are characterized by the increased expression of IFN-regulatory genes, implicating dysregulation of the innate immune response in activation of these cells. However, what triggers and sustains monocyte activation in SSc remains unclear. Since Epstein-Barr virus (EBV) mRNA and proteins were found in fibroblasts and endothelial cells in SSc skin, we sought to determine whether EBV might also infect monocytes and contribute to their activation in SSc.

Methods:

Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of CD19+ cell fraction, using CD14/CD16 negative depletion (CD14-) (Human Monocyte Enrichment Kit without CD16 Depletion,EasySep, StemCell). Circulating monocytes from diffuse cutaneous SSc (dcSSc) (n=8) and healthy donors (HDs) (n=6), were examined for the presence of EBV lytic proteins using Immunofluorescence (IF). EBV-p2089 recombinant virus was used to infect dcSSc and HDs monocytes. Gene expression of IFNs, TLRs (TLR7/8/9) and innate immune mediators (IRF5/7) was evaluated in EBV-p2089 infected dcSSc and HD monocytes using real-time PCR, and proteins examined by IF staining and Western Blot. Flow cytometry was performed on dcSSc and HD PBMCs labeled with phycoerythrin (PE), allophycocyanin (APC), fluorescein isothiocynate (FITC) and PE-cyanine7 (PE-Cy7) conjugated mouse monoclonal antibodies (mAb) against human CD14, CD16, CD163, CD206, CD169/siglec1, CD4, CD8, CD20, CD19, CD23. THP1 monocytes were stimulated with TLR7 (R837) and TLR8 synthetic ligands (cpd14b, R848).

Results:

We found that EBV lytic proteins (Zebra, BFRF1 and gp-350/220), were expressed in skin macrophages and in circulating monocytes from SSc patients, while no expression of lytic EBV was detected in monocytes/macrophages from HDs. Infection of SSc monocytes by EBV-p2089 strongly induced TLR8 expression, while no induction of TLR7 and TLR9 was observed in the infected cells. EBV also significantly induced markers of activated monocytes, such as IRF7, IRF5, Siglec1 and IL-6. Further supporting the importance of TLR8 activation in SSc, expression of TLR8 was significantly increased in freshly isolated monocytes from dcSSc patients compared to HDs. Furthermore, distinct monocyte subsets (CD14+/CD16++) and activation markers were identified in dcSSc PBMCs compared to HDs by FACS analysis. Activation of TLR8 by synthetic ligands mimicked EBV effects on TLR8, inducing IRF7 and inflammatory cytokines, whereas the TLR7 agonist did not induce monocyte inflammation markers on THP1 cells.

Conclusion: These data suggest that dcSSc monocytes are carrying an EBV lytic infection. Activation of TLR8 by EBV RNA might represent a novel mechanism in mediating monocyte inflammation in SSc by which EBV triggers the innate immune response in infected cells.

Disclosure: A. Farina, None; G. Peruzzi, None; V. Lacconi, None; E. Rosato, None; S. Quarta, None; S. Morrone, None; A. Faggioni, None; M. Trojanowska, None; G. A. Farina, None.

To cite this abstract in AMA style:

Farina A, Peruzzi G, Lacconi V, Rosato E, Quarta S, Morrone S, Faggioni A, Trojanowska M, Farina GA. Epstein-Barr Virus Induces Activation of Infammatory Markers Via the TLR8 Transduction Pathway in Infected Scleroderma Monocytes [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/epstein-barr-virus-induces-activation-of-infammatory-markers-via-the-tlr8-transduction-pathway-in-infected-scleroderma-monocytes/. Accessed .

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