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Cerebral organoids exhibit microglia, astrocyte, and neuronal cells and express HIV receptors. (A) Double immunostainings of S100b (astrocyte), IBA1 (microglia), and Tuj1 (neurons) combined with nuclear staining Hoechst after 8 weeks in culture. Representative pictures of cerebral organoids from iPSC line OH1.5 are shown. Magnification of 20× and 40× was used. (B) mRNA expression levels of microglia, astrocyte, and neuron-specific markers and HIV receptors were assessed by qRT-PCR. Gene expression was normalized to the reference gene ACTB. The means ± standard errors of the means are shown. (C–E) SORT-seq data of LPS-stimulated organoids after 9-10 weeks in culture. (C) Annotated clusters on tSNE plot. Cluster 5 (C5) was only partially annotated. Cluster 9 (C9) was not annotated. (D) Heatmap of Patir et al. (2019) microglia signature gene expression across each cluster. (E) Violin plots of CD4, CCR5, and CXCR4 gene expression. Expression levels shown are log-normalized and covariate corrected.
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Abstract: The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV’s entry into the CNS, latency establishment, persistence, and reversal. Therefore, re...
Citations
... Without diminishing the importance of these advances in vascularization technology, we note that if the hypothesis evaluated does not require culturing for three months, then vascularization of the organoid may not be necessary. For example, a study of HIV infection in microglia-containing human brain organoids found that the organoids were generally suitable for research on infection-related questions if they were five to nine weeks old (Gumbs et al. 2022). ...
Most current information about neurological disorders and diseases is derived from direct patient and animal studies. However, patient studies in many cases do not allow replication of the early stages of the disease and, therefore, offer limited opportunities to understand disease progression. On the other hand, although the use of animal models allows us to study the mechanisms of the disease, they present significant limitations in developing drugs for humans. Recently, 3D-cultured in vitro models derived from human pluripotent stem cells have surfaced as a promising system. They offer the potential to connect findings from patient studies with those from animal models. In this comprehensive review, we discuss their application in modeling neurodevelopmental conditions such as Down Syndrome or Autism, neurodegenerative diseases such as Alzheimer’s or Parkinson’s, and viral diseases like Zika virus or HIV. Furthermore, we will discuss the different models used to study prenatal exposure to drugs of abuse, as well as the limitations and challenges that must be met to transform the landscape of research on human brain disorders.
Graphical Abstract
... In the context of immune cell-associated genes, there were also increases in the genes that encode T cell CD8α and CD8β chains in 5XFAD/MR1 KO mice, whereas CD4 gene expression was decreased in these mice. The expression changes are across all cell populations; therefore, it is important to note that CD4 can be upregulated on microglia during an human immunodeficiency virus infection 22 and CD8 has been found on microglia/macrophages after a stroke. 23 How this might contribute to the reduced pathology in the MR1-deficient 5XFAD mice remains to be determined. ...
INTRODUCTION
Plaques are a hallmark feature of Alzheimer's disease (AD). We found that the loss of mucosal‐associated invariant T (MAIT) cells and their antigen‐presenting molecule MR1 caused a delay in plaque pathology development in AD mouse models. However, it remains unknown how this axis is impacting dystrophic neurites.
METHODS
Brain tissue from 5XFAD mice and those that are MR1 deficient (MR1 KO), were analyzed for dystrophic neurites, amyloid plaques, and synapses via immunofluorescence, RNA sequencing, enzyme‐linked immunosorbent assay, and western blot.
RESULTS
In 8‐month‐old 5XFAD/MR1 KO mice, there was reduced expression of lysosomal‐associated membrane protein 1, ubiquitin, and n‐terminal amyloid precursor protein in the hippocampus compared to 5XFAD mice (P < 0.05). 5XFAD/MR1 KO mice also had less insoluble amyloid beta 40 (P < 0.001) and higher levels of postsynaptic density protein 95 (P < 0.01) in the hippocampus.
DISCUSSION
Our data contribute additional mechanistic insight into the detrimental role of the MR1/MAIT cell axis in AD pathology development.
Highlights
5XFAD mice lacking the innate immune MR1/MAIT (mucosal‐associated invariant T) cell axis (5XFAD/MR1 KO) have reduced numbers of dystrophic neurite markers in the hippocampus at 8 months of age.
Hippocampal tissue transcriptional analyses showed reduced expression of genes encoding classical dystrophic neurite markers in 5XFAD/MR1 KO mice.
5XFAD/MR1 KO mice had less insoluble amyloid beta 40 and increased levels of the post‐synaptic marker, postsynaptic density protein 95, in the hippocampus than did MR1+ 5XFAD mice.
... Organoids are one in vitro model that is currently being used to study HIV infection of the CNS [185][186][187]. Organoids are 3D miniatures of the organ that are made using stem cell progenitors and are being widely used explore neuroinflammations and neurodegeneration [188]. ...
Although treatable with antiretroviral therapy, HIV infection persists in people living with HIV (PLWH). It is well known that the HIV virus finds refuge in places for which antiretroviral medications do not reach therapeutic levels, mainly the CNS. It is clear that as PLWH age, the likelihood of developing HIV-associated neurological deficits increases. At the biochemical level neurological dysfunction is the manifestation of altered cellular function and ineffective intercellular communication. In this review, we examine how intercellular signaling in the brain is disrupted in the context of HIV. Specifically, the concept of how the blood-brain barrier can be a convergence point for crosstalk, is explored. Crosstalk between the cells of the neurovascular unit (NVU) (endothelium, pericytes, astrocytes, microglia and neurons) is critical for maintaining proper brain function. In fact, the NVU allows for rapid matching of neuronal metabolic needs, regulation of blood-brain barrier (BBB) dynamics for nutrient transport and changes to the level of immunosurveillance. This review invites the reader to conceptually consider the BBB as a router or convergence point for NVU crosstalk, to facilitate a better understanding of the intricate signaling events that underpin the function of the NVU during HIV associated neuropathology.
... Though valuable, most preclinical studies of HIV-1 brain infection currently rely on either small animal models that only express a subset of viral proteins, humanized mice that do not fully reconstitute human CNS cells and architecture, or SIV-infected macaques, which do not fully reflect HIV-associated neurocognitive pathologies [51][52][53][54]. Some HIV-1 studies take advantage of brain organoids made of human CNS cells [55][56][57], but these systems fail to recapitulate the structure of the human brain and lack its cellular diversity [58]. Furthermore, they require addition of microglia from an external source or derive microglia from induced pluripotent stem cells [59], which is an important caveat since microglia remain as HIV reservoirs in people taking antiretroviral therapies [60]. ...
HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.
... CD4 was mainly expressed in microglia while CXCR4 was detected in in a much broader array of cell types (Fig. 4B). These results are consistent with findings recently reported by Gumb et al. (37). To examine changes in gene expression occurring in microglia infected with HIV, we enriched these cells using CD11B+ microbeads. ...
Despite the success of combination antiretroviral therapy (ART) for individuals living with HIV, mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia forms the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation, neuronal dysfunction and/or death observed in HAND. Utilizing two different iPSC-derived brain organoid models (cerebral and choroid plexus (ChP) organoids) containing microglia, we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1, ISG15, ISG20, IFI27 and IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART, consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by scRNA-seq. However, S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells, mature and immature ChP cells, neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons, accompanied by increased expression of genes promoting cellular senescence and cell death. Together, these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons.
... One of the most challenging aspects of HAND is the latent pool of infected cells that persist during cART. The development of cellular, three-dimensional organoid, and small animal models of HIV-1 infection in microglia has greatly improved our molecular understanding of how intermittent emergence of HIV-1 from latency in microglial cells primarily contributes to both neuroinflammation in the CNS and the progression of HAND [40, [120][121][122][123][124]. ...
Despite combined antiretroviral therapy (cART) limiting HIV replication to undetectable levels in the blood, people living with HIV continue to experience HIV-associated neurocognitive disorder (HAND). HAND is associated with neurocognitive impairment, including motor impairment, and memory loss. HIV has been detected in the brain within 8 days of estimated exposure and the mechanisms for this early entry are being actively studied. Once having entered into the central nervous system (CNS), HIV degrades the blood–brain barrier through the production of its gp120 and Tat proteins. These proteins are directly toxic to endothelial cells and neurons, and propagate inflammatory cytokines by the activation of immune cells and dysregulation of tight junction proteins. The BBB breakdown is associated with the progression of neurocognitive disease. One of the main hurdles for treatment for HAND is the latent pool of cells, which are insensitive to cART and prolong inflammation by harboring the provirus in long-lived cells that can reactivate, causing damage. Multiple strategies are being studied to combat the latent pool and HAND; however, clinically, these approaches have been insufficient and require further revisions. The goal of this paper is to aggregate the known mechanisms and challenges associated with HAND.
... Though valuable, most preclinical studies of HIV-1 brain infection currently rely on either small animal models that only express a subset of viral proteins, humanized mice that don't fully reconstitute human CNS cells and architecture, or SIVinfected macaques, which don't fully reflect HIV-associated neurocognitive pathologies [49][50][51][52]. Some HIV-1 studies take advantage of brain organoids made of human CNS cells [53][54][55], but these systems fail to recapitulate the structure of the human brain and lack its cellular diversity [56]. Further, they require addition of microglia from an external source or derive microglia from induced pluripotent stem cells [57], which is an important caveat since microglia remain as HIV reservoirs in people taking antiretroviral therapies [58]. ...
HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissoci-ated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9-days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular via-bility over time. Overall, this human-centered model offers a promising resource to study the cel-lular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.
... As a result, there is the phosphorylation of molecules related to the activation of the NF-kB pathway, which release, in the nucleus, molecules related to the subsequent increased release of CCL5/RANTES, correlated with the increase in neuroinflammation and cellular damages in the brain [62]. The main cells of the CNS affected by neuroAIDS, as described in the literary scene, are: neurons; microglia; astrocytes; and oligodendrocytes [32,40,42,[51][52][53]56,60,66]. These tissue injuries cause clinical problems in affected individuals [67]. ...
Chronic HIV-1 infection can cause neurological illness, also known as HIV-associated neurocognitive disorders (HAND). The elevated level of pro-inflammatory cytokines and chemokines, such as C-C Chemokine Ligand 5 (CCL5/RANTES), is one of the ways of causing HIV-1-mediated neuroinflammation. C-C Chemokine Receptor 5 (CCR5) is the main coreceptor for viral entry into host cells and for mediating induction of CCL5/RANTES. CCR5 and CCL5 are part of a correlated axis of immune pathways used for effective protection against the HIV-1 virus. The purpose of this paper was to review the literary knowledge about the immunopathological relationship between this immune complex and neuroAIDS. A systematic review of the literature was conducted based on the selection and search of articles, available in English, Spanish, or Portuguese in the time frame of 1990–2022, of primary and secondary types in the PUBMED, Science Direct, SciELO, and LILACS databases through descriptors (MeSH) together with “AND”: “CCR5”; “CCL5”; “neurological manifestations”; or “HIV”. The methodological quality of the articles was assessed using the JBI Checklists and the PRISMA 2020 writing guidelines were followed. A total of 36 articles were included in the final composition of the review. The main cells of the CNS affected by neuroAIDS are: neurons; microglia; astrocytes; and oligodendrocytes. Molecular devices and their associations with cellular injuries have been described from the entry of the virus into the host’s CNS cell to the generation of mental disorders. Furthermore, divergent results were found about the levels of CCL5/RANTES secretion and the generation of immunopathogenesis, while all condensed research for CCR5 indicated that elevation of this receptor causes more neurodegenerative manifestations. Therefore, new therapeutic and interventional strategies can be conditioned on the immunological direction proposed in this review for the disease.
... Recently, cerebral organoids have emerged as a significant tool for investigating neurotropic viruses, in particular HIV (Swingler et al. 2023), even with limited studies using human brain organoids as a model for HIV infection have been reported (dos Reis et al. 2020;Gumbs et al. 2022). The first model of 3D human brain organoids (hBORG) with integrated microglia (MG) was developed by dos Reis et al. (dos Reis et al. 2020;Dos Reis et al. 2023). ...
... Indeed, the importance of microglia stems from their essential role, alongside macrophages, as major cell types accountable for HIV infection and replication within the brain (Wiley et al. 1999). More recently, Gumbs et al. described a model of microglia-containing cerebral organoids, which they successfully infected with HIV (Gumbs et al. 2022). The model was derived from iPSCs and showed presence of cells from the three germ layers at initial stages of organoid generation (Ormel et al. 2018). ...
The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38 million people worldwide with nearly 1.6 million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.
... As such, it was widely thought that cerebral organoids did not contain microglia and would not produce interferon response upon viral infection. However, earlier studies had discovered and characterized CD11b+ microglia that differentiated innately within these cerebral organoids [47][48][49][50] . ...
Neuroinflammation is a central process in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease (AD), and there are active efforts to target pathways involved in neuroinflammation for molecular biomarker discovery and therapeutic development in neurodegenerative diseases. It was also proposed that there may be an infectious etiology in AD that is associated with viruses such as herpes simplex virus (HSV-1) and influenza A virus (IAV), leading to neuroinflammation-induced AD pathogenesis or disease progression. We sought to develop high-throughput, quantitative molecular biomarker assays using dissociated cells from human cerebral organoids (dcOrgs), that can used for screening compounds to reverse AD-associated neuroinflammation. We found that HSV-1 infection, but not IAV infection, in dcOrgs led to increased intracellular Aβ42 and phosphorylated Tau-Thr212 (pTau-212) expression, lower ratios of secreted Aβ42/40, as well as neuronal loss, and increased proportions of astrocytes and microglia, which are hallmarks of AD. Among the glia cell-type markers, Iba1 (microglia) and GFAP (astrocyte) expression were most strongly correlated with HSV-1 expression, which further supported that these biomarkers are perturbed by glia-mediated neuroinflammation. By performing large-scale RNA sequencing, we observed that differentially expressed transcripts in HSV-1 infected dcOrgs were specifically enriched for AD-associated GWAS genes, but not for genes associated with other common neurodegenerative, neuropsychiatric or autoimmune diseases. Immediate treatment of HSV-1 infected dcOrgs with anti-herpetic drug acyclovir (ACV) rescued most of the cellular and transcriptomic biomarkers in a dosage-dependent manner, indicating that it is possible to use our high-throughput platform to identify compounds or target genes that can reverse these neuroinflammation-induced biomarkers associated with AD.
