Cellular immune responses in acute hepatitis E virus infection to the viral open reading frame 2 protein

Abstract

Hepatitis E virus (HEV) causes acute viral hepatitis and is endemic in the developing world. Few data are available on cellular immune responses in HEV infection. Using flow cytometry, we studied the frequencies of peripheral blood CD4(+) /CD8(+) T cells secreting interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-4 in 21 patients with acute hepatitis E and 18 healthy controls, after stimulation with the HEV capsid (ORF2) protein. Cytokine levels in serum specimens and culture supernatants of ORF2-stimulated peripheral blood mononuclear cells (PBMCs) were estimated in enzyme-linked immunosorbent assays. In addition, cytokine mRNA transcripts were measured in PBMCs by reverse transcription-polymerase chain reaction. In patients with acute hepatitis E, although the total CD4(+) population was expanded, the proportions of CD4(+)/CD69(+) and CD8(+) /CD69(+) cells producing IFN-gamma, TNF-alpha, and IL-4 in response to HEV ORF2 stimulation were unchanged. However, IFN-gamma levels in the supernatants and IFN-gamma mRNA transcripts in cells were elevated in ORF2-stimulated PBMCs in acute hepatitis E; levels of IL-2 or TNF-alpha were unchanged. Our findings suggest that CD4(+) IFN-gamma-secreting cells, which do not belong either to the helper T cell type 1 or type 2 phenotype, as is the case with natural killer T cells, may be involved in the pathogenesis of hepatitis E. Further, the limited immune reactivity we detected in peripheral blood cells may be related to the sequestration of immune events to the intrahepatic compartment, which is the major disease site.

FIG. 1

Representative dot plots of three-color fluorescence data for detection of cells staining for intracellular cytokines for one healthy control subject (A) and one patient (B). Both (A) and (B) show results after stimulation of whole blood with PMA and ionomycin (top row) or hepatitis E virus ORF2 protein (middle and bottom rows) for 6 h with addition of BD GolgiStop (BD Biosciences Pharmingen) for the last 5 h of culture. Cells were surface stained with fluorescein isothiocyanate (FITC)-labeled antibodies to CD3, CD4, or CD8; fixed; permeabilized; and then labeled with PerCP-labeled CD69 and one phycoerythrin (PE)-labeled anti-cytokine (IFN-α, TNF-α, or IL-2) antibody. The gate for mononuclear cells was drawn using forward scatter (FSC) and side scatter (SSC) parameters, and 20,000 events were acquired within this gate. Cells positive for CD3 (top row), CD4 (middle row), and CD8 (bottom row) were gated on FL-1 and SSC (first column). Within each gate, the proportion of CD69- and cytokine-positive cells was analyzed; the cutoffs used were based on isotype control for the CD3⁺ population (top row) and the corresponding unstimulated cells for the CD4⁺ and CD8⁺ cells (middle and bottom rows).

FIG. 1

Representative dot plots of three-color fluorescence data for detection of cells staining for intracellular cytokines for one healthy control subject (A) and one patient (B). Both (A) and (B) show results after stimulation of whole blood with PMA and ionomycin (top row) or hepatitis E virus ORF2 protein (middle and bottom rows) for 6 h with addition of BD GolgiStop (BD Biosciences Pharmingen) for the last 5 h of culture. Cells were surface stained with fluorescein isothiocyanate (FITC)-labeled antibodies to CD3, CD4, or CD8; fixed; permeabilized; and then labeled with PerCP-labeled CD69 and one phycoerythrin (PE)-labeled anti-cytokine (IFN-α, TNF-α, or IL-2) antibody. The gate for mononuclear cells was drawn using forward scatter (FSC) and side scatter (SSC) parameters, and 20,000 events were acquired within this gate. Cells positive for CD3 (top row), CD4 (middle row), and CD8 (bottom row) were gated on FL-1 and SSC (first column). Within each gate, the proportion of CD69- and cytokine-positive cells was analyzed; the cutoffs used were based on isotype control for the CD3⁺ population (top row) and the corresponding unstimulated cells for the CD4⁺ and CD8⁺ cells (middle and bottom rows).

FIG. 2

Representative cytokine mRNA expression in peripheral blood mononuclear cells obtained from one patient with acute hepatitis E and one healthy control subject. Lane 1, unstimulated; lane 2, after stimulation with phytohemagglutinin (PHA); lane 3, after stimulation with HEV ORF2 protein.

FIG. 3

Interleukin-1β levels in serum samples from patients with acute hepatitis E and from controls.