Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells

Abstract

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Glycolytic requirement for NK cell cytotoxicity and cytomegalovirus control

“…2DG treatment was associated with higher viral burdens as early as day 2 after infection in the spleen ( Figure 4A), and by day 4, there was significant loss of viral control, evidenced by high viral copy number in both the spleen and liver ( Figure 4B). Data on organ-specific control of MCMV is mixed, but one hypothesis is that NK cell IFN-γ is more important for control of MCMV in the liver, while NK cell cytotoxicity and Ly49H are responsible for control of infection in the spleen (44)(45)(46)(47), which is consistent with our findings.…”

“…Tissue pieces (5 mm 3 ) were minced and placed in Gentra Puregene Cell Lysis Buffer (Qiagen), and DNA was extracted following the manufacturer's recommended protocol and diluted to 20-50ng/ml. MCMV immediate-early gene 1-specific (ie1-specific) primers and probe were used to determine viral copy number as described (47) and normalized to β-actin (Actb). Reactions were run in duplicate on a 7500 Fast Real-Time PCR instrument (Applied Biosystems) with an absolute standard curve.…”

Chronic alcohol consumption exacerbates murine cytomegalovirus infection via impairing nonspecific and specific NK activation in mice

“…Deficiency in IFN‐α/β or NK/IFN‐γ dramatically enhances viral replication . The antiviral potency of IFN‐β is approximately 30 times stronger than IFN‐α . Chronic alcohol consumption significantly decreased blood level IFN‐β and the IFN‐γ‐producing NK cells at 12 hours pi (Figure ), which would enhance MCMV replication and dissemination leading to the increase in viral load in the spleen as we observed 4 days pi (Figure ).…”

“…IFN‐γ and perforin are the two most important effector molecules that control viral replication and clearance . IFN‐γ plays a key role in the control of viral replication .…”

“…Spleen and liver are the two organs that are rapidly infected and most severely damaged after CMV infection . Multiple types of cytokines and immune cells are involved in the anti‐CMV immune response and among those IL‐12, IFN‐α/β, IFN‐γ, and IL‐15 are the most important cytokines with NK cells and CD8 + T cells being important immune effector cells that control viral replication and clearance . NK cells play a pivotal role in the control of CMV infection.…”