Multiscale imaging of HIV-1 transmission in humanized mice

HIV transmission within lymphoid tissues remains incompletely characterized at the level of individual cells and virions. Here we visually describe our approach to understanding HIV-1 dissemination at different levels of volume and resolution within lymphoid tissues from HIV-1-infected humanized mice. We combined tissue clearing techniques, immunostaining, and light sheet fluorescence microscopy to visualize large-volumes of intact tissue with single-cell resolution from HIV-1-infected humanized mice. In parallel, we imaged adjacent regions of tissue using electron microscopy and electron tomography to gain 3D ultrastructural information about the same tissue samples. This approach can provide spatial information about the density and distribution of target cells, HIV-1-infected cells, and individual budding and free-virions within lymphoid tissues. Multiscale imaging of HIV-1 infected tissues from humanized mice can provide insight into the biological mechanisms of HIV-1 transmission through the correlation of global pathology with structural details and these methods are directly translatable to other animal models and human clinical samples

Multiscale Imaging of HIV-1 Transmission in Humanized Mice

HIV transmission within lymphoid tissues remains incompletely characterized at the level of individual cells and virions. Here we visually describe our approach to understanding HIV-1 dissemination at different levels of volume and resolution within lymphoid tissues from HIV-1-infected humanized mice. We combined tissue clearing techniques, immunostaining, and light sheet fluorescence microscopy to visualize large-volumes of intact tissue with single-cell resolution from HIV-1-infected humanized mice. In parallel, we imaged adjacent regions of tissue using electron microscopy and electron tomography to gain 3D ultrastructural information about the same tissue samples. This approach can provide spatial information about the density and distribution of target cells, HIV-1-infected cells, and individual budding and free-virions within lymphoid tissues. Multiscale imaging of HIV-1 infected tissues from humanized mice can provide insight into the biological mechanisms of HIV-1 transmission through the correlation of global pathology with structural details and these methods are directly translatable to other animal models and human clinical samples

In vitro characterization of engineered red blood cells as viral traps against HIV-1 and SARS-CoV-2

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps, as RBCs lack nuclei and other organelles required for viral replication. However, expression of viral receptors on RBCs is difficult to achieve since mature erythrocytes lack the cellular machinery to synthesize proteins. Herein, we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein in erythroid progenitor cells, which efficiently differentiated into enucleated RBCs. HIV-1 efficiently entered RBCs that co-expressed CD4 and CCR5, but viral entry was not required for neutralization, as CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 and prevent infection of CD4+ T cells in vitro due to the formation of high-avidity interactions with trimeric HIV-1 Env spikes on virions. To facilitate continuous large-scale production of RBC viral traps, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our in vitro results suggest that this approach warrants further investigation as a potential treatment against acute and chronic viral infections

Multiscale imaging of HIV-1 transmission in humanized mice

HIV transmission within lymphoid tissues remains incompletely characterized at the level of individual cells and virions. Here we visually describe our approach to understanding HIV-1 dissemination at different levels of volume and resolution within lymphoid tissues from HIV-1-infected humanized mice. We combined tissue clearing techniques, immunostaining, and light sheet fluorescence microscopy to visualize large-volumes of intact tissue with single-cell resolution from HIV-1-infected humanized mice. In parallel, we imaged adjacent regions of tissue using electron microscopy and electron tomography to gain 3D ultrastructural information about the same tissue samples. This approach can provide spatial information about the density and distribution of target cells, HIV-1-infected cells, and individual budding and free-virions within lymphoid tissues. Multiscale imaging of HIV-1 infected tissues from humanized mice can provide insight into the biological mechanisms of HIV-1 transmission through the correlation of global pathology with structural details and these methods are directly translatable to other animal models and human clinical samples

Electron Tomography of HIV-1 Infection in Gut-Associated Lymphoid Tissue

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1–infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1–infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue

Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography

Dissemination of HIV-1 throughout lymphoid tissues leads to systemic virus spread following infection. We combined tissue clearing, 3D-immunofluorescence, and electron tomography (ET) to longitudinally assess early HIV-1 spread in lymphoid tissues in humanized mice. Immunofluorescence revealed peak infection density in gut at 10-12 days post-infection when blood viral loads were low. Human CD4+ T-cells and HIV-1-infected cells localized predominantly to crypts and the lower third of intestinal villi. Free virions and infected cells were not readily detectable by ET at 5-days post-infection, whereas HIV-1-infected cells surrounded by pools of free virions were present in ~10% of intestinal crypts by 10-12 days. ET of spleen revealed thousands of virions released by individual cells and discreet cytoplasmic densities near sites of prolific virus production. These studies highlight the importance of multiscale imaging of HIV-1-infected tissues and are adaptable to other animal models and human patient samples

Dioxin Exposure Blocks Lactation Through a Direct Effect on Mammary Epithelial Cells Mediated by the Aryl Hydrocarbon Receptor Repressor

In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene β-casein. Since TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition