Caspase cleavage of gasdermin E causes neuronal pyroptosis in HIV-associated neurocognitive disorder.

Despite effective antiretroviral therapies, 20-30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with ß-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders.

Gasdermin D activation in oligodendrocytes and microglia drives inflammatory demyelination in progressive multiple sclerosis.

Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.

Microbial molecule ingress promotes neuroinflammation and brain CCR5 expression in persons with HIV-associated neurocognitive disorders.

BACKGROUND: Systemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. METHODS: Brain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. FINDINGS: Among NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250-550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. INTERPRETATION: Increased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

Mutant KRAS regulates transposable element RNA and innate immunity via KRAB zinc-finger genes.

RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway.

Functional and structural segregation of overlapping helices in HIV-1.

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.

A high-quality bonobo genome refines the analysis of hominid evolution.

The divergence of chimpanzee and bonobo provides one of the few examples of recent hominid speciation1,2. Here we describe a fully annotated, high-quality bonobo genome assembly, which was constructed without guidance from reference genomes by applying a multiplatform genomics approach. We generate a bonobo genome assembly in which more than 98% of genes are completely annotated and 99% of the gaps are closed, including the resolution of about half of the segmental duplications and almost all of the full-length mobile elements. We compare the bonobo genome to those of other great apes1,3-5 and identify more than 5,569 fixed structural variants that specifically distinguish the bonobo and chimpanzee lineages. We focus on genes that have been lost, changed in structure or expanded in the last few million years of bonobo evolution. We produce a high-resolution map of incomplete lineage sorting and estimate that around 5.1% of the human genome is genetically closer to chimpanzee or bonobo and that more than 36.5% of the genome shows incomplete lineage sorting if we consider a deeper phylogeny including gorilla and orangutan. We also show that 26% of the segments of incomplete lineage sorting between human and chimpanzee or human and bonobo are non-randomly distributed and that genes within these clustered segments show significant excess of amino acid replacement compared to the rest of the genome.

Intranasal anti-caspase-1 therapy preserves myelin and glucose metabolism in a model of progressive multiple sclerosis.

Inflammatory demyelination and axonal injury in the central nervous system (CNS) are cardinal features of progressive multiple sclerosis (MS), and linked to activated brain macrophage-like cells (BMCs) including resident microglia and trafficking macrophages. Caspase-1 is a pivotal mediator of inflammation and cell death in the CNS. We investigated the effects of caspase-1 activation and its regulation in models of MS. Brains from progressive MS and non-MS patients, as well as cultured human oligodendrocytes were examined by transcriptomic and morphological methods. Next generation transcriptional sequencing of progressive MS compared to non-MS patients' normal appearing white matter (NAWM) showed induction of caspase-1 as well as other inflammasome-associated genes with concurrent suppression of neuron-specific genes. Oligodendrocytes exposed to TNFα exhibited upregulation of caspase-1 with myelin gene suppression in a cell differentiation state-dependent manner. Brains from cuprizone-exposed mice treated by intranasal delivery of the caspase-1 inhibitor, VX-765 or its vehicle, were investigated in morphological and molecular studies, as well as by fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. Cuprizone exposure resulted in BMC and caspase-1 activation accompanied by demyelination and axonal injury, which was abrogated by intranasal VX-765 treatment. FDG-PET imaging revealed suppressed glucose metabolism in the thalamus, hippocampus and cortex of cuprizone-exposed mice that was restored with VX-765 treatment. These studies highlight the caspase-1 dependent interactions between inflammation, demyelination, and glucose metabolism in progressive MS and associated models. Intranasal delivery of an anti-caspase-1 therapy represents a promising therapeutic approach for progressive MS and other neuro-inflammatory diseases.

The UCSC Genome Browser database: 2021 update.

For more than two decades, the UCSC Genome Browser database (https://genome.ucsc.edu) has provided high-quality genomics data visualization and genome annotations to the research community. As the field of genomics grows and more data become available, new modes of display are required to accommodate new technologies. New features released this past year include a Hi-C heatmap display, a phased family trio display for VCF files, and various track visualization improvements. Striving to keep data up-to-date, new updates to gene annotations include GENCODE Genes, NCBI RefSeq Genes, and Ensembl Genes. New data tracks added for human and mouse genomes include the ENCODE registry of candidate cis-regulatory elements, promoters from the Eukaryotic Promoter Database, and NCBI RefSeq Select and Matched Annotation from NCBI and EMBL-EBI (MANE). Within weeks of learning about the outbreak of coronavirus, UCSC released a genome browser, with detailed annotation tracks, for the SARS-CoV-2 RNA reference assembly.

Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility.

The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

Stability of SARS-CoV-2 phylogenies.

The SARS-CoV-2 pandemic has led to unprecedented, nearly real-time genetic tracing due to the rapid community sequencing response. Researchers immediately leveraged these data to infer the evolutionary relationships among viral samples and to study key biological questions, including whether host viral genome editing and recombination are features of SARS-CoV-2 evolution. This global sequencing effort is inherently decentralized and must rely on data collected by many labs using a wide variety of molecular and bioinformatic techniques. There is thus a strong possibility that systematic errors associated with lab-or protocol-specific practices affect some sequences in the repositories. We find that some recurrent mutations in reported SARS-CoV-2 genome sequences have been observed predominantly or exclusively by single labs, co-localize with commonly used primer binding sites and are more likely to affect the protein-coding sequences than other similarly recurrent mutations. We show that their inclusion can affect phylogenetic inference on scales relevant to local lineage tracing, and make it appear as though there has been an excess of recurrent mutation or recombination among viral lineages. We suggest how samples can be screened and problematic variants removed, and we plan to regularly inform the scientific community with our updated results as more SARS-CoV-2 genome sequences are shared (https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473 and https://virological.org/t/masking-strategies-for-sars-cov-2-alignments/480). We also develop tools for comparing and visualizing differences among very large phylogenies and we show that consistent clade- and tree-based comparisons can be made between phylogenies produced by different groups. These will facilitate evolutionary inferences and comparisons among phylogenies produced for a wide array of purposes. Building on the SARS-CoV-2 Genome Browser at UCSC, we present a toolkit to compare, analyze and combine SARS-CoV-2 phylogenies, find and remove potential sequencing errors and establish a widely shared, stable clade structure for a more accurate scientific inference and discourse.

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis.

BACKGROUND: Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation. METHODS: A siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semi-quantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG35-55-induced EAE, and in matched controls. RESULTS: Among progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1ß release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia. CONCLUSIONS: Activation of executioner caspases-3/7, widely considered key mediators of apoptosis, contributed to GSDMD-associated microglial pyroptosis under neuroinflammatory conditions. Collectively, these observations highlight the convergence of different cell death pathways during neuroinflammation and offer new therapeutic opportunities in neuroinflammatory disease.

Co-option of the lineage-specific LAVA retrotransposon in the gibbon genome.

Co-option of transposable elements (TEs) to become part of existing or new enhancers is an important mechanism for evolution of gene regulation. However, contributions of lineage-specific TE insertions to recent regulatory adaptations remain poorly understood. Gibbons present a suitable model to study these contributions as they have evolved a lineage-specific TE called LAVA (LINE-AluSz-VNTR-AluLIKE), which is still active in the gibbon genome. The LAVA retrotransposon is thought to have played a role in the emergence of the highly rearranged structure of the gibbon genome by disrupting transcription of cell cycle genes. In this study, we investigated whether LAVA may have also contributed to the evolution of gene regulation by adopting enhancer function. We characterized fixed and polymorphic LAVA insertions across multiple gibbons and found 96 LAVA elements overlapping enhancer chromatin states. Moreover, LAVA was enriched in multiple transcription factor binding motifs, was bound by an important transcription factor (PU.1), and was associated with higher levels of gene expression in cis We found gibbon-specific signatures of purifying/positive selection at 27 LAVA insertions. Two of these insertions were fixed in the gibbon lineage and overlapped with enhancer chromatin states, representing putative co-opted LAVA enhancers. These putative enhancers were located within genes encoding SETD2 and RAD9A, two proteins that facilitate accurate repair of DNA double-strand breaks and prevent chromosomal rearrangement mutations. Co-option of LAVA in these genes may have influenced regulation of processes that preserve genome integrity. Our findings highlight the importance of considering lineage-specific TEs in studying evolution of gene regulatory elements.

A survey-based analysis of the academic job market.

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success - including funding, number of publications or journals published in - were unable to completely differentiate applicants with and without job offers. Respondents also reported that the hiring process was unnecessarily stressful, time-consuming, and lacking in feedback, irrespective of outcome. Our findings suggest that there is considerable scope to improve the transparency of the hiring process.

The UCSC repeat browser allows discovery and visualization of evolutionary conflict across repeat families.

BACKGROUND: Nearly half the human genome consists of repeat elements, most of which are retrotransposons, and many of which play important biological roles. However repeat elements pose several unique challenges to current bioinformatic analyses and visualization tools, as short repeat sequences can map to multiple genomic loci resulting in their misclassification and misinterpretation. In fact, sequence data mapping to repeat elements are often discarded from analysis pipelines. Therefore, there is a continued need for standardized tools and techniques to interpret genomic data of repeats. RESULTS: We present the UCSC Repeat Browser, which consists of a complete set of human repeat reference sequences derived from annotations made by the commonly used program RepeatMasker. The UCSC Repeat Browser also provides an alignment from the human genome to these references, uses it to map the standard human genome annotation tracks, and presents all of them as a comprehensive interface to facilitate work with repetitive elements. It also provides processed tracks of multiple publicly available datasets of particular interest to the repeat community, including ChIP-seq datasets for KRAB Zinc Finger Proteins (KZNFs) - a family of proteins known to bind and repress certain classes of repeats. We used the UCSC Repeat Browser in combination with these datasets, as well as RepeatMasker annotations in several non-human primates, to trace the independent trajectories of species-specific evolutionary battles between LINE 1 retroelements and their repressors. Furthermore, we document at https://repeatbrowser.ucsc.edu how researchers can map their own human genome annotations to these reference repeat sequences. CONCLUSIONS: The UCSC Repeat Browser allows easy and intuitive visualization of genomic data on consensus repeat elements, circumventing the problem of multi-mapping, in which sequencing reads of repeat elements map to multiple locations on the human genome. By developing a reference consensus, multiple datasets and annotation tracks can easily be overlaid to reveal complex evolutionary histories of repeats in a single interactive window. Specifically, we use this approach to retrace the history of several primate specific LINE-1 families across apes, and discover several species-specific routes of evolution that correlate with the emergence and binding of KZNFs.

Breast Tumor-Associated Metalloproteases Restrict Reovirus Oncolysis by Cleaving the σ1 Cell Attachment Protein and Can Be Overcome by Mutation of σ1.

Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyomavirus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein σ1, which diminished virus binding to sialic acid (SA)-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated σ1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231, and HS578T breast cancer cell lines recapitulated σ1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers and that breast cancer cells by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of SA levels on a panel of cancer cells showed that truncated σ1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in σ1 that prevents σ1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, toxicity equivalent to that of wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data show that tumor microenvironments have the potential to reduce infectivity of reovirus.IMPORTANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have a negative impact on proteinaceous cancer therapies, such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

Monitoring Breast Cancer Response to Neoadjuvant Chemotherapy Using Ultrasound Strain Elastography.

Strain elastography was used to monitor response to neoadjuvant chemotherapy (NAC) in 92 patients with biopsy-proven, locally advanced breast cancer. Strain elastography data were collected before, during, and after NAC. Relative changes in tumor strain ratio (SR) were calculated over time, and responder status was classified according to tumor size changes. Statistical analyses determined the significance of changes in SR over time and between response groups. Machine learning techniques, such as a naïve Bayes classifier, were used to evaluate the performance of the SR as a marker for Miller-Payne pathological endpoints. With pathological complete response (pCR) as an endpoint, a significant difference (P < .01) in the SR was observed between response groups as early as 2 weeks into NAC. Naïve Bayes classifiers predicted pCR with a sensitivity of 84%, specificity of 85%, and area under the curve of 81% at the preoperative scan. This study demonstrates that strain elastography may be predictive of NAC response in locally advanced breast cancer as early as 2 weeks into treatment, with high sensitivity and specificity, granting it the potential to be used for active monitoring of tumor response to chemotherapy.

Highly Mutable Linker Regions Regulate HIV-1 Rev Function and Stability.

HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev's established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev's ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein.

IL-23 signaling in Th17 cells is inhibited by HIV infection and is not restored by HAART: Implications for persistent immune activation.

OBJECTIVES: HIV infection causes a profound depletion of gut derived Th17 cells, contributing to loss of mucosal barrier function and an increase in microbial translocation, thus driving systemic immune activation. Despite normalization of circulating CD4+ T cell counts with highly active antiretroviral therapy (HAART), Th17 frequency and function often remain impaired. Given the importance of interleukin (IL)-23 in the generation and stabilization of Th17 cells we hypothesized that impaired IL-23 signaling causes persistent Th17 dysfunction in HIV infection. METHODS: The effects of in vitro HIV infection on responses to IL-23 in Th17 cells were examined. These included the production of IL-17, phosphorylated STAT3 (pSTAT3) and the transcription of retinoic acid orphan receptor C (RORC) gene. Blood derived Th17 cells from untreated and HAART-treated HIV-infected individuals were also examined for the IL-23 induced production of phosphorylated STAT3 (pSTAT3) and the expression of the IL-23 receptors. RESULTS: In vitro HIV infection significantly inhibited IL-17 production and IL-23 induced pSTAT3 while expression of RORC RNA was unaffected. Th17 cells isolated from untreated and HAART-treated HIV-infected individuals showed complete loss of IL-23 induced pSTAT3 without a decrease in the expression of the IL-23 receptors. CONCLUSIONS: This study is the first to demonstrate an effect of HIV on the IL-23 signaling pathway in Th17 cells. We show that in vitro and in vivo HIV infection results in impaired IL-23 signaling which is not reversed by HAART nor is it a result of reduced receptor expression, suggesting that HIV interferes with IL-23-activated signaling pathways. These findings may explain the inability of HAART to restore Th17 frequency and function and the resulting persistent chronic immune activation observed in HIV infected individuals.

HIV and HIV-Tat inhibit LPS-induced IL-27 production in human macrophages by distinct intracellular signaling pathways.

Monocyte-derived Mϕs (MDMs) from HIV-infected patients and MDM infected in vitro with HIV exhibit a reduced ability to secrete various cytokines, including IL-12. Recently, IL-27, an IL-12 family cytokine, was shown to inhibit HIV replication in Mϕ. Whether HIV infection or HIV accessory protein(s) impact IL-27 production in Mϕs remains unknown. Herein, we show that in vitro HIV infection, as well as intracellular HIV-Tat (Tat) and Tat peptides, inhibit LPS-induced IL-27 production in human MDMs, suggesting impairment of the TLR4 signaling pathway. To understand the signaling pathways governing HIV or Tat-mediated inhibition of LPS-induced IL-27 production, we first demonstrated that p38 MAPK, PI3K, Src-homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1), and Src kinases regulate LPS-induced IL-27 production in MDMs. Tat caused down-regulation of TNFR-associated factor (TRAF)-6 and inhibitor of apoptosis 1 (cIAP-1) and subsequently decreased phosphorylation of downstream PI3K and p38 MAPKs, which were implicated in LPS-induced IL-27 production. Whereas SHP-1 and Src kinases regulated LPS-induced IL-27 production, Tat did not inhibit these kinases, suggesting that they were not involved in Tat-mediated inhibition of LPS-induced IL-27 production. In contrast to Tat, in vitro HIV infection of MDM inhibited LPS-induced IL-27 production via inhibition of p38 MAPK activation. Overall, HIV and Tat inhibit LPS-induced IL-27 production in human macrophages via distinct mechanisms: Tat through the inhibition of cIAP-1-TRAF-6 and subsequent inhibition of PI3K and p38 MAPKs, whereas HIV through the inhibition of p38 MAPK activation.