Cell death and cell proliferation in mouse submandibular gland during early post-irradiation phase.

The effects of irradiation on different cell compartments in the submandibular gland were analyzed in adult C57BL/6 mice exposed to X-ray irradiation and followed up for 10 days. Apoptosis was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method (TUNEL). Cell proliferation was detected using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Radiation-induced apoptosis occurred rapidly, reaching a maximum 3 days post-irradiation. The percentage of apoptotic cells increased with the irradiation dose. At day 1 post-irradiation, cell proliferation was significantly reduced in comparison to sham-irradiated controls. After post-irradiation arrest of the cell cycle, proliferation increased in all gland compartments, reaching a maximum at day 6 post-irradiation. The proliferation response corresponded to the dose of irradiation. We suggest that the reason for gland dysfunction could be the coexistence of high apoptotic and proliferative activity in the irradiated gland.</p

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Treg cells are crucial for immune homeostasis and for dampening immune response to several diseased conditions, including viral infections. Murine cytomegalovirus (MCMV) is a herpesvirus with pathogenic potential, so that early immune mechanisms are essential in controlling virus and protecting from virus-induced pathology. Studies on Foxp3+ Treg cells have revealed their inhibitory role on the early T cell response to MCMV infection and have suggested Treg cells as a target of MCMV’s immunoevasion mechanisms. Here we demonstrate that the number and activation status of liver Treg cells is strongly induced upon MCMV infection in order to protect the host from severe liver damage. They constitutively express high amounts of IL-33 receptor ST2 and their accumulation depends on IL-33, which is released as a tissue alarmin after the cell damage. For the first time, we show an immunoregulatory role of IL-33-dependent Treg cells in the liver of MCMV infected mice and their suppression of MCMV-induced immunopathology

Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand.

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, theattenuatedgrowth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell– based vaccine

CD4 T cells are required for maintenance of CD8 TRM cells and virus control in the brain of MCMV-infected newborn mice

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8+ T cells, while CD4+ T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here, we set out to determine the role of CD4+ T cells upon MCMV infection of newborn mice. We provide evidence that CD4+ T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4+ T cells are required for induction and maintenance of tissue- resident memory CD8+ T cells in the brain of mice perinatally infected with MCMV

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

<div><p>Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.</p></div

Persistent expression of proinflammatory chemokines in cochlea of mice with ABR thresholds >60dB.

<p>Mice were infected with MCMV or mock infected as controls as described in Materials and Methods. One group of infected mice (n = 12) and control mice (n = 6) were harvested at PNd10. A second group underwent ABR testing on PNd42 and equal numbers (n = 12) of mice with ABR thresholds >60dB (median 75dB) or <60dB (median 45dB) were harvested, cochlea pooled from individual mice and expression of proinflammatory chemokines determined as described in Materials and Methods. Results are reported as fold change and statistical comparison to age matched control mice (p value). Note increased expression of CCL8, CXCL9, and CXCL10 in mice with hearing loss.</p><p>Persistent expression of proinflammatory chemokines in cochlea of mice with ABR thresholds >60dB.</p

Loss of spiral ganglion neurons in mice infected in newborn period with MCMV.

<p>Mice were sacrificed at various postnatal days (PNd) 7, 11, and 30 as described in the Material and Methods. For each time point, cochlea were removed and placed in 4% paraformaldehyde, decalcified and embedded in paraffin as described in Materials and Methods. Sections were stained with anti-Tuji 1 antibodies to detect neurons followed by horseradish peroxidase (HRP) conjugated second antibody and development with diaminobenzidine (brown). Cresyl violet (CV) was used as counterstain. Digitalized images, of the inner ear from each individual tsection (2 per cochlea) were imported into the <i>B-Cell Image</i> program. Spiral ganglion cell (SGC) densities were calculated in infected animals (4 mice per time point) and mock infected animals (3 mice per time point) by determining the number of spiral ganglion neurons followed by normalization to the area of Rosenthal’s canal in each section to provide the spiral ganglion neuron density (cells/mm²). Spiral ganglion neuron densities in the basal, mid and apical turns (I, II, III) of the cochlea are presented as the mean number of neuron density with error bars demarcating SEM (<b>**</b> indicates significant differences p<0.0022, and *** p<0.001 by Mann-Whitney).</p

Progressive hearing loss and virus inoculum effect on progression of hearing loss in MCMV infected mice.

<p><b>(A)</b> MCMV infected mice exhibit increased ABR threshold as function of age. Newborn mice infected with uncloned Smith MCMV(●; n = 12) or mock infected (<b>○;</b> n = 15) were tested at approximately 3,7, and 12 months and ABR thresholds determined. Note increasing mean ABR threshold in infected mice at 7 months of age and increase in ABR threshold for both infected and mock infected groups of mice at 12 months of age. At each time point the mean ABR thresholds of mock vs. infected mice were significantly different (p<0.001 two-way Anova with Bonferroni post-test correction). (<b>B)</b> Dose dependence of progressive hearing loss and delayed onset of hearing loss. Newborn mice were inoculated with recombinant, BAC cloned MCMV(50PFU ●; 200PFU ▲) or mock uninfected (<b>○</b>) and tested for auditory function at PNd42 or PNd78. Each symbol represents one ear tested (n = 96 mock, n = 36(50PFU), n = 52(200PFU)). Note the increase in median ABR threshold in mice given 50 PFU between PNd42 and PNd78 as compared to mock infected animals. (<b>**</b>) indicates significant differences (p<0.01) between groups with increase in ABR thresholds as determined by Kruskal-Wallis test of medians with Dunn post-test correction. <b>(C)</b> Magnitude of ABR thresholds in cochlea with progression of hearing loss. The change (dB) in individual cochlea exhibiting an increase in ABR threshold between d42 and d78 (Δ ABR threshold) was plotted for each group and medians compared. Number of cochlea with progression that were analyzed were (mock = 27; 50PFU = 13; 200PFU = 26). Statistically significant differences between median Δ ABR threshold of mock vs 50PFU, (p<0.01) and mock vs. 200PFU (p<0.001) by Kruskal-Wallis test of medians with Dunn post-test correction.</p