An Efficient Humanized Mouse Model for Oral Anti-Retroviral Administration.

HIV anti-retrovirals (ARVs) have vastly improved the life expectancy of people living with HIV (PLWH). However, toxic effects attributed to long-term ARV use also contribute to HIV-related co-morbidities such as heart disease, bone loss and HIV-associated neurocognitive disorders (HAND). Unfortunately, mouse models used to study the effects of ARVs on viral suppression, toxicity and HIV latency/tissue reservoirs have not been widely established. Here, we demonstrate an effective mouse model utilizing immune-compromised mice, reconstituted with infected human peripheral blood mononuclear cell (PBMCs). ARVs areincorporated into mouse chow and administered daily with combination ARV regimens includingAtripla (efavirenz, tenofovir disoproxil fumarate, and emtricitabine) and Triumeq (abacavir, dolutegravir and lamivudine). This model measures HIV-infected human cell trafficking, and ARV penetration throughout most relevant HIV organs and plasma, with a large amount of trafficking to the secondary lymphoid organs. Furthermore, the HIV viral load within each organ and the plasma was reduced in ARV treated vs. untreated control. Overall, we have demonstrated a mouse model that is relatively easy and affordable to establish and utilize to study ARVs' effect on various tissues, including the co-morbid conditions associated with PLWH, such as HAND, and other toxic effects.

ß-catenin regulates HIV latency and modulates HIV reactivation.

Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, ß-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the ß-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. ß-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of ß-catenin and each LRA class tested. In sum, we identify ß-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts.

ß-Catenin Restricts Zika Virus Internalization by Downregulating Axl.

The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, ß-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited ß-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that ß-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for ß-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and ß-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that ß-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates ß-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits ß-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

Migration of CD8+ T Cells into the Central Nervous System Gives Rise to Highly Potent Anti-HIV CD4dimCD8bright T Cells in a Wnt Signaling-Dependent Manner.

The role of CD8(+) T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8(+) T cells dimly express CD4 on their surface. This population is known as CD4(dim)CD8(bright) T cells. We evaluated the role of CD4(dim)CD8(bright) and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ(-/-) mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4(dim)CD8(bright)) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4(dim)CD8(bright) T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4(dim)CD8(bright) T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4(dim)CD8(bright) T cells from HIV-infected mice exhibited anti-HIV-specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide-loaded targets. Further, higher frequency of CD4(dim)CD8(bright) T cells (R = -0.62; p ≤ 0.001), but not CD8 single positive T cells (R = -0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8(+) T cells entering the CNS during HIV infection can give rise to CD4(dim)CD8(bright) T cells, likely through a Wnt signaling-dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4(dim)CD8(bright) T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.

Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS.

HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either ß-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5-fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.

ß-Catenin/TCF-4 signaling regulates susceptibility of macrophages and resistance of monocytes to HIV-1 productive infection.

Cells of the monocyte/macrophage lineage are an important target for HIV-1 infection. They are often at anatomical sites linked to HIV-1 transmission and are an important vehicle for disseminating HIV-1 throughout the body, including the central nervous system. Monocytes do not support extensive productive HIV-1 replication, but they become more susceptible to HIV-1infection as they differentiate into macrophages. The mechanisms guiding susceptibility of HIV-1 replication in monocytes versus macrophages are not entirely clear. We determined whether endogenous activity of ß-catenin signaling impacts differential susceptibility of monocytes and monocyte-derived macrophages (MDMs) to productive HIV-1 replication. We show that monocytes have an approximately 4-fold higher activity of ß-catenin signaling than MDMs. Inducing ß-catenin in MDMs suppressed HIV-1 replication by 5-fold while inhibiting endogenous ß-catenin signaling in monocytes by transfecting with a dominant negative mutant for the downstream effector of ß- catenin (TCF-4) promoted productive HIV-1 replication by 6-fold. These findings indicate that ß-catenin/TCF-4 is an important pathway for restricted HIV-1 replication in monocytes and plays a significant role in potentiating HIV-1 replication as monocytes differentiate into macrophages. Targeting this pathway may provide a novel strategy to purge the latent reservoir from monocytes/macrophages, especially in sanctuary sites for HIV-1 such as the central nervous system.

Porcupine is not required for the production of the majority of Wnts from primary human astrocytes and CD8+ T cells.

Wnts are small secreted glycoproteins that are highly conserved among species. To date, 19 Wnts have been described, which initiate a signal transduction cascade that is either ß-catenin dependent or independent, culminating in the regulation of hundreds of target genes. Extracellular release of Wnts is dependent on lipidation of Wnts by porcupine, a membrane-bound-O-acyltransferase protein in the endoplasmic reticulum. Studies demonstrating the requirement of porcupine for Wnts production are based on cell line and non-human primary cells. We evaluated the requirement for porcupine for Wnts production in human primary astrocytes and CD8+ T cells. Using IWP-2, an inhibitor of porcupine, or siRNA targeting porcupine, we demonstrate that porcupine is not required for the release of Wnt 1, 3, 5b, 6,7a, 10b, and 16a. While IWP had no effect on Wnt 2b release, knockdown of porcupine by siRNA reduced Wnt 2b release by 60%. These data indicate that porcupine-mediated production of Wnts is context dependent and is not required for all Wnts production, suggesting that alternative mechanisms exist for Wnts production.