EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities
Brandon S. Cox, Khaled Alharshawi, Irene Mena-Palomo, William P. Lafuse, Maria Eugenia Ariza
Brandon S. Cox, Khaled Alharshawi, Irene Mena-Palomo, William P. Lafuse, Maria Eugenia Ariza
View: Text | PDF
Research Article Infectious disease

EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities

Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A–dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.

Authors

Brandon S. Cox, Khaled Alharshawi, Irene Mena-Palomo, William P. Lafuse, Maria Eugenia Ariza

×

Figure 7

EBV dUTPase injections induce TFH cell differentiation and the formation of NKTFH cells in spleens of C57BL/6 female mice.

Options: View larger image (or click on image) Download as PowerPoint
EBV dUTPase injections induce TFH cell differentiation and the formation...
(A) Spleen single-cell suspensions (splenocytes) from EBV dUTPase– or vehicle-injected mice for 5 consecutive days were prepared as described in Methods. Cells were immediately stained with Abs against CD4, IL-21, BCL6, CD19 (B cells), CD11b (monocytes), and CD11c (DC), and the expression of TFH markers IL-21 and BCL6 was examined by flow cytometry analysis. Flow cytometry analysis was conducted on the lymphocyte population and pregated on live (FVD–) CD19–CD11b–CD11c– cells. Representative flow cytometry plots indicating frequency (%) of CD4+IL-21+ cells in gate (left) or BCL6+ T cells (right) in EBV dUTPase– and vehicle control–injected mice. (B and C) Splenocytes (prepared following injections as in A) were depleted of CD19+ B cells and CD8+ T cells by magnetic cell separation using MojoSort nanobeads, as described in Methods. Enriched cells were stained with Abs against B220, CD11b, CD11c, CD4, CD3, CD1d-PBS-57 tetramer, and CXCR5, and the expression of tetramer subsets was analyzed by flow cytometry. Flow cytometry samples were pregated on live (FVD–) B220–CD11b–CD11c– cells. (B) Flow cytometry of splenic CD4+ CD1d-PBS-57 tetramer–negative T cells (gating), indicating percentage of CXCR5+ TFH cells. (C) Flow cytometry and relative frequencies of CD1d-PBS-57 tetramer–positive CD3int NKT cells in gate (left plots) or CXCR5hi NKTFH cells (right plots). Bar graph represents quantitative data of NKTFH cells in mice. Data represent the mean ± SEM of 3 independent experiments; n = 20 (10 mice/group) (A), n = 10 (5 mice/group) (B). *P < 0.05, ***P < 0.001, by 2-tailed Mann-Whitney U test (A–C).