Spinal Cord Disease in HIV Infection

HIV infection is associated with numerous spinal cord diseases, such as vacuolar myelopathy, primary HIV-associated acute transverse myelitis, and primary CNS lymphoma, amongst others. These diseases had a much higher prevalence in the pre-cART era, however, some individuals are still affected despite cART treatment. Moreover, a previous study has shown that HIV-1 gp120 induces synaptic degeneration in the spinal pain neural circuit, which is likely a critical step in neuropathogenesis of the spinal cord in HIV. Further study is needed to better understand how HIV patients are affected by spinal cord disease, and to develop therapeutic strategies. To study HIV in its relation to spinal cord disease, we used a recently developed humanized mouse model that has human microglial cell reconstitution. This model allows for the HIV infection in central nervous system and the observation of resulting pathology.https://digitalcommons.unmc.edu/surp2021/1057/thumbnail.jp

Preliminary Studies on Immune Response and Viral Pathogenesis of Zika Virus in Rhesus Macaques

Zika Virus (ZIKV) is primarily transmitted through mosquito bites. It can also be transmitted during sexual intercourse and in utero from mother to fetus. To gain preliminary insight into ZIKV pathology and immune responses on route of transmission, rhesus macaques (RMs) were inoculated with ZIKV (PRVABC59) via intravaginal (IVAG) (n = 3) or subcutaneous (sub Q) (n = 2) routes. Systemic ZIKV infection was observed in all RMs, regardless of the route of inoculation. After 9 days postinfection (dpi), ZIKV was not detected in the plasma of IVAG- and sub-Q-inoculated RMs. Importantly, RMs harbored ZIKV up to 60 dpi in various anatomical locations. Of note, ZIKV was also present in several regions of the brain, including the caudate nucleus, parietal lobe, cortex, and amygdala. These observations appear to indicate that ZIKV infection may be systemic and persistent regardless of route of inoculation. In addition, we observed changes in key immune cell populations in response to ZIKV infection. Importantly, IVAG ZIKV infection of RMs is associated with increased depletion of CD11C hi myeloid cells, reduced PD-1 expression in NK cells, and elevated frequencies of Ki67⁺ CD8⁺ central memory cells as compared to sub Q ZIKV-infected RMs. These results need to interpreted with caution due to the small number of animals utilized in this study. Future studies involving large groups of animals that have been inoculated through both routes of transmission are needed to confirm our findings

Human-like NSG Mouse Glycoproteins Sialylation Pattern Changes the Phenotype of Human Lymphocytes and Sensitivity to HIV-1 Infection

BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins\u27 chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγ CONCLUSION: NSG-cma

Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice

Human immunodeficiency virus type one (HIV-1) tissue compartments are established soon after viral infection. However, the timing in which virus gains a permanent foothold in tissue and the cellular factors that control early viral-immune events are incompletely understood. These are critical events in studies of HIV-1 pathogenesis and in the development of viral reservoirs after antiretroviral therapy. Moreover, factors affecting the permanence of viral-tissue interactions underlie barriers designed to eliminate HIV-1 infection. To this end we investigated the temporal and spatial viral and host factors during HIV-1 seeding of tissue compartments. Two humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mouse models were employed. In the first, immune deficient mice were reconstituted with human CD34+ cord blood hematopoietic stem cells (HSC) (hu-HSC) and in the second mice were transplanted with adult mature human peripheral lymphocytes (hu-PBL). Both, in measure, reflect relationships between immune activation and viral infection as seen in an infected human host. Following humanization both mice models were infected with HIV-1ADA at 104 50% tissue culture infective doses. Viral nucleic acids and protein and immune cell profiles were assayed in brain, lung, spleen, liver, kidney, lymph nodes, bone marrow, and gut from 3 to 42 days. Peripheral CD4+ T cell loss began at 3 days together with detection of HIV-1 RNA in both mouse models after initiation of HIV-1 infection. HIV-1 was observed in all tested tissues at days 3 and 14 in hu- PBL and HSC mice, respectively. Immune impairment was most prominent in hu-PBL mice. T cell maturation and inflammation factors were linked directly to viral tissue seeding in both mouse models. We conclude that early viral tissue compartmentalization provides a roadmap for investigations into HIV-1 elimination

In vivo confocal microscopic analysis of normal human anterior limbal stroma.

PURPOSE: To characterize the microarchitecture of anterior limbal stroma in healthy individuals using in vivo confocal microscopy (IVCM) and to correlate it with mesenchymal stem cells (MSCs), a component of the limbal niche. METHODS: The corneal side of the superior limbus was scanned in 30 eyes of 17 normal subjects beyond the basal epithelium, deep into the stroma using an HRT III laser scanning microscope. The IVCM findings were correlated with the immunohistochemical features of MSCs in the anterior limbal stroma. RESULTS: Clusters of hyperreflective structures were observed in the anterior limbal stroma, subjacent to the basal epithelium (depth, 50.2 ± 8.7 μm to 98 ± 12.8 μm), but not in the corneal stroma. The structures showed unique morphology compared with epithelial cells, keratocytes, neurons, and dendritic cells. In parallel, confocal analysis of immunostained sections showed clusters of cells, double positive for MSC-specific markers (CD90 and CD105) in the anterior limbal stroma at a depth of 55.3 ± 12.7 μm to 72 ± 37.6 μm. The organization and distribution of the MSC clusters locates them within the hyperreflective region in the anterior limbal stroma. CONCLUSIONS: The hyperreflective structures, demonstrated for the first time in the human anterior limbal stroma, probably represent an important component of the limbal niche. Our approach of in vivo imaging may pave the way for assessing the limbal stromal health

Human Interleukin-34 facilitates microglia-like cell differentiation and persistent HIV-1 infection in humanized mice

Abstract Background Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell’s presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration. Methods Herein, we created a humanized bone-marrow chimera producing human “microglia like” cells in NOD.Cg-Prkdc scid Il2rg tm1Sug Tg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain. Results Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses. Conclusions This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain

Preliminary Studies on Immune Response and Viral Pathogenesis of Zika Virus in Rhesus Macaques

Zika Virus (ZIKV) is primarily transmitted through mosquito bites. It can also be transmitted during sexual intercourse and in utero from mother to fetus. To gain preliminary insight into ZIKV pathology and immune responses on route of transmission, rhesus macaques (RMs) were inoculated with ZIKV (PRVABC59) via intravaginal (IVAG) (n = 3) or subcutaneous (sub Q) (n = 2) routes. Systemic ZIKV infection was observed in all RMs, regardless of the route of inoculation. After 9 days postinfection (dpi), ZIKV was not detected in the plasma of IVAG- and sub-Q-inoculated RMs. Importantly, RMs harbored ZIKV up to 60 dpi in various anatomical locations. Of note, ZIKV was also present in several regions of the brain, including the caudate nucleus, parietal lobe, cortex, and amygdala. These observations appear to indicate that ZIKV infection may be systemic and persistent regardless of route of inoculation. In addition, we observed changes in key immune cell populations in response to ZIKV infection. Importantly, IVAG ZIKV infection of RMs is associated with increased depletion of CD11C hi myeloid cells, reduced PD-1 expression in NK cells, and elevated frequencies of Ki67+ CD8+ central memory cells as compared to sub Q ZIKV-infected RMs. These results need to interpreted with caution due to the small number of animals utilized in this study. Future studies involving large groups of animals that have been inoculated through both routes of transmission are needed to confirm our findings

Influence of Notch signaling on p27^kip1 and Skp2 expressions.

<p><b>(A, B)</b> Activation of Notch signaling (Jag1) significantly decreased and increased levels of <i>p27</i>^<i>kip1</i> (a) and <i>Skp2</i> (b) transcripts, respectively, compared to controls. Their levels were recovered to that in controls in the presence of DAPT. <b>(C)</b> Schematic representation of <i>p27</i>^<i>kip1</i> proximal promoter containing an evolutionarily conserved C-box (C-site) element. <b>(D)</b> ChIP analysis carried out on enriched MG using Hes1 antibody revealed a sequence corresponding to <i>Hes1</i> promoter containing the C-site, as demonstrated by the size and sequence specific PCR products. <b>(E)</b> Schematic representation of <i>Skp2</i> proximal promoter containing evolutionarily conserved CSL binding sites. <b>(F)</b> ChIP analysis carried out on enriched MG using CSL antibody revealed sequences corresponding to CSL promoter containing the CSL sites, as demonstrated by the size and sequence specific PCR products. The specificity of the ChIP assays was demonstrated by the absence of PCR products in the IgG controls. *p<0.05. Data are mean ± SEM.</p

Post-translational influence of Notch signaling on p27^Kip1 and Skp2 expression.

<p><b>(A)</b> Western analyses revealed robust levels of p27^Kip1 in enriched MG, which relatively decreased in FGF2/Jag-1 groups and increased in cells treated with DAPT. <b>(B)</b> In contrast, relative to that of p27^Kip1, levels of Skp2 were less in MG but increased in cells treated with FGF2/Jag-1 and decreased in the DAPT group. <b>(C)</b> An increase and a decrease in protein levels of NICD were observed in the presence of FGF2/Jag1 and DAPT, respectively, indicating that Notch signaling was perturbed. Levels in graphs represent the arbitrary scanning units and all protein levels were normalized to GAPDH. <b>(D, E)</b> Quantitative Western analysis revealed a significant decrease and increase in levels of p27^Kip1 (d) and Skp2 (e) protein levels in Jag1-treated groups, respectively, versus controls (FGF2), confirming the qualitative analyses carried out on the pooled samples (seen in “A”). *p<0.05. Data are mean ± SEM.</p

Notch signaling regulates p27^Kip1 expression to influence the activation of MG.

<p>p27^Kip1 expression persists in MG as one of their characteristic molecular features. The activation of Notch signaling in quiescent MG leads to a decrease in the expression of p27^Kip1 through Hes1-mediated transcriptional repression and Skp2-mediated post-translational inhibition, promoting the G1-S transition.</p