VZV Infection of Primary Human Adrenal Cortical Cells Produces a Proinflammatory Environment without Cell Death

Abstract

Virus infection of adrenal glands can disrupt secretion of mineralocorticoids, glucocorticoids, and sex hormones from the cortex and catecholamines from the medulla, leading to a constellation of symptoms such as fatigue, dizziness, weight loss, nausea, and muscle and joint pain. Specifically, varicella zoster virus (VZV) can produce bilateral adrenal hemorrhage and adrenal insufficiency during primary infection or following reactivation. However, the mechanisms by which VZV affects the adrenal glands are not well-characterized. Herein, we determined if primary human adrenal cortical cells (HAdCCs) infected with VZV support viral replication and produce a proinflammatory environment. Quantitative PCR showed VZV DNA increasing over time in HAdCCs, yet no cell death was seen at 3 days post-infection by TUNEL staining or Western Blot analysis with PARP and caspase 9 antibodies. Compared to conditioned supernatant from mock-infected cells, supernatant from VZV-infected cells contained significantly elevated IL-6, IL-8, IL-12p70, IL-13, IL-4, and TNF-α. Overall, VZV can productively infect adrenal cortical cells in the absence of cell death, suggesting that these cells may be a potential reservoir for ongoing viral replication and proinflammatory cytokine production, leading to chronic adrenalitis and dysfunction.

Keywords: adrenal cortex; adrenal glands; adrenalitis; cytokines; inflammation; varicella zoster virus.

Figure 1

VZV productively infects quiescent human adrenal cortical cells (qHAdCCs). (A) At 3 days post-infection (DPI), VZV-infected qHAdCCs had a cytopathic effect that was absent in mock-infected cells on phase-contrast microscopy. (B) VZV DNA significantly increased over time at 1, 2, and 3 DPI; VZV DNA was absent in mock-infected cells at 3 DPI. (C) In VZV-infected qHAdCCs, adrenal cortical cell purity was confirmed by expression of glial fibrillary acidic protein (GFAP, left panel), but not tyrosine hydroxylase (TH; middle panel); cells expressed VZV glycoprotein B (gB, right panel), indicating productive virus infection in this cell type. Image magnification A = 4×, C= 10×. DAPI = nuclei dye; N.D. = not detected. * = p < 0.05, ** = p < 0.01.

Figure 2

VZV infection of quiescent human adrenal cortical cells (qHAdCCs) does not cause cell death. (A) At 3 days post-infection (DPI), a TUNEL assay kit with BrdU-Red that detects DNA fragmentation during cell death (apoptosis, necroptosis, and pyroptosis) did not show increased staining in VZV- compared to mock-infected qHAdCCs. (B) Western blot analysis was completed to assess for hallmarks of apoptosis (cleaved caspase 9 [Cas-9] and cleaved poly [ADP-ribos] polymerase [PARP]). The apoptosis negative control was uninfected human lung fibroblasts (HLFs, lane 2) and the positive control was HLFs treated with 1µM saurosporine to induce apoptosis (lane 3). The negative and positive apoptosis controls, mock-infected qHAdCCs (lanes 4 and 5, duplicates), and VZV-infected qHAdCCs (lanes 7 and 8, duplicates) all contained bands corresponding to full-length PARP (F-PARP, 116 kDa); cleaved PARP (C-PARP, 89 kDa) was only present in the positive control; C-PARP was absent in the negative control and mock- and VZV-infected cells. The apoptosis controls (negative and positive), mock-infected qHAdCCs, and VZV-infected qHAdCCs all contained bands corresponding to full-length caspase 9 (F-Cas9, 47 kDa). Cleaved caspase 9 (C-Cas9, 37 kDa) was seen in the positive control and absent or barely detectable in the negative control and mock- and VZV-infected cells. Blots were stripped and probed for β-actin as a loading control. L = Molecular weight ladder. Image magnification A = 10×.

Figure 3

VZV-infected quiescent human adrenal cortical cells (qHAdCCs) secrete proinflammatory cytokines. (A) At 3 days post-infection, conditioned supernatant from mock- and VZV-infected cells were analyzed for proinflammatory cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13, IFN-γ, and TNF-α by a multiplex assay. Compared to mock-infected cells, VZV-infected cells had significantly increased levels of IL-6 (p = 0.0005) and IL-8 (p = 0.0002). IL-12p70, IL-13, IL-4, and TNF-α were detected in supernatant from VZV-infected cells, but not from mock-infected cells. (B) As a positive control to demonstrate that qHAdCCs have the capacity to secrete proinflammatory cytokines, qHAdCCs were treated with lipopolysaccharide (LPS) for 24 h. Compared to Mock qHAdCCs, LPS-treated cells showed significantly elevated levels of IL-6 (p < 0.0001) and IL-8 (p < 0.0001); IL-10, IL-12p70, IL-13, IL-1β, IL-4, and TNF-α were also present in LPS-treated cell supernatant but not detected in mock. N.D. = not detected. **** = p < 0.0001, *** = p < 0.0005.