Myeloid cell tropism enables MHC-E-restricted CD8+ T cell priming and vaccine efficacy by the RhCMV/SIV vaccine.

The strain 68-1 rhesus cytomegalovirus (RhCMV)-based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8+ T cells that recognize epitopes presented by major histocompatibility complex (MHC)-II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II- and/or MHC-Ia-restricted CD8+ T cells do not protect against SIV, it remains unclear whether MHC-E-restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II-restricted component. Using host microRNA (miR)-mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope-targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142-mediated tropism restriction eliminated MHC-E epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-II epitope-targeted response. Inhibition with the endothelial cell-selective miR-126 eliminated MHC-II epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-E epitope-targeted response. Dual miR-142 + miR-126-mediated tropism restriction reverted CD8+ T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8+ T cell responses were generally similar, only the vectors programmed to elicit MHC-E-restricted CD8+ T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion.

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

Evaluation of the molecular mechanisms associated with cytotoxicity and inflammation after pulmonary exposure to different metal-rich welding particles.

Welding generates a complex aerosol of incidental nanoparticles and cytotoxic metals, such as chromium (Cr), manganese (Mn), nickel (Ni), and iron (Fe). The goal was to use both in vivo and in vitro methodologies to determine the mechanisms by which different welding fumes may damage the lungs. Sprague-Dawley rats were treated by intratracheal instillation (ITI) with 2.0 mg of gas metal arc-mild steel (GMA-MS) or manual metal arc-stainless steel (MMA-SS) fumes or saline (vehicle control). At 1, 3, and 10 days, bronchoalveolar lavage (BAL) was performed to measure lung toxicity. To assess molecular mechanisms of cytotoxicity, RAW264.7 cells were exposed to both welding fumes for 24 h (0-100 µg/ml). Fume composition was different: MMA-SS (41% Fe, 29% Cr, 17% Mn, 3% Ni) versus GMA-MS (85% Fe, 14% Mn). BAL indicators of lung injury and inflammation were increased by MMA-SS at all time points and by GMA-MS at 3 and 10 days after exposure. RAW264.7 cells exposed to MMA-SS had elevated generation of reactive oxygen species (ROS), protein-HNE (P-HNE) adduct formation, activation of ERK1/2, and expression of cyclooxygenase-2 (COX-2) compared to GMA-MS and control. Increased generation of ROS due to MMA-SS exposure was confirmed by increased expression of Nrf2 and heme oxygenase-1 (HO-1). Results of in vitro studies provide evidence that stainless steel welding fume mediate inflammatory responses via activation of ROS/P-HNE/ERK1/2/Nrf2 signaling pathways. These findings were corroborated by elevated expression of COX-2, Nrf2, and HO-1 in homogenized lung tissue collected 1 day after in vivo exposure.

Oxidative Stress, DNA Methylation, and Telomere Length Changes in Peripheral Blood Mononuclear Cells after Pulmonary Exposure to Metal-Rich Welding Nanoparticles.

Welding fume is a complex mixture of different potentially cytotoxic and genotoxic metals, such as chromium (Cr), manganese (Mn), nickel (Ni), and iron (Fe). Documented health effects have been observed in workers exposed to welding fume. The objective of the study was to use an animal model to identify potential biomarkers of epigenetic changes (e.g., changes in telomere length, DNA methylation) in isolated peripheral blood mononuclear cells (PBMCs) after exposure to different welding fumes. Male Sprague-Dawley rats were exposed by intratracheal instillation (ITI) of 2.0 mg/rat of gas metal arc-mild steel (GMA-MS) or manual metal arc-stainless steel (MMA-SS) welding fume. Vehicle controls received sterile saline by ITI. At 4 h, 14 h, 1 d, 3 d, 10 d, and 30 d, bronchoalveolar lavage (BAL) was performed to assess lung inflammation. Whole blood was collected, and PBMCs were isolated. Dihydroethidium (DHE) fluorescence and 4-hydroxylnonenal protein adduct (P-HNE) formation were measured in PBMCs to assess reactive oxygen species (ROS) production. DNA alterations in PBMCs were determined by evaluating changes in DNA methylation and telomere length. Metal composition of the two fumes was different: MMA-SS (41 % Fe, 29 % Cr, 17 % Mn, 3 % Ni) versus GMA-MS (85 % Fe, 14 % Mn). The more soluble and chemically complex MMA-SS sample induced a more persistent and greater inflammatory response compared to the other groups. Also, oxidative stress markers increased at 24 h in the PBMCs recovered from the MMA-SS group compared to other group. No significant differences were observed when comparing DNA methylation between the welding fume and control groups at any of the time points, whereas the MMA-SS sample significantly increased telomere length at 1 and 30 d after a single exposure compared to the other groups. These findings suggest that genotoxic metals in MMA-SS fume (e.g., Cr and Ni), that are absent in the GMA-MS fume, may enhance lung toxicity, as well as induce markers of oxidative stress and increase telomere length in PBMCs. Importantly, the measurement of telomere length in cells isolated from peripheral blood may serve as a potential biomarker of response in the assessment of toxicity associated with welding fumes.