Endogenous Retroviruses Research Articles

SAMHD1 Inhibitor Combined with Immune Checkpoint Inhibitor Elicits an Anti-AML Immunity

Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

Deletion of Vβ3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vβ3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vβ3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

Roles of Human Endogenous Retroviruses and Endogenous Virus-Like Elements in Cancer Development and Innate Immunity.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections in the host genome. Although mutations and silencing mechanisms impair their original role in viral replication, HERVs are believed to play roles in various biological processes. Long interspersed nuclear elements (LINEs) are non-LTR retrotransposons that have a lifecycle resembling that of retroviruses. Although LINE expression is typically silenced in somatic cells, it also contributes to various biological processes. The aberrant expression of HERVs and LINEs is closely associated with the development of cancer and/or immunological diseases, suggesting that they are integrated into various pathways related to the diseases. HERVs/LINEs control gene expression depending on the context as promoter/enhancer elements. Some RNAs and proteins derived from HERVs/LINEs have oncogenic potential, whereas others stimulate innate immunity. Non-retroviral endogenous viral elements (nrEVEs) are a novel type of virus-like element in the genome. nrEVEs may also be involved in host immunity. This article provides a current understanding of how these elements impact cellular physiology in cancer development and innate immunity, and provides perspectives for future studies.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Captive ERVWE1 triggers impairment of 5-HT neuronal plasticity in the first-episode schizophrenia by post-transcriptional activation of HTR1B in ALKBH5-m6A dependent epigenetic mechanisms

BackgroundAbnormalities in the 5-HT system and synaptic plasticity are hallmark features of schizophrenia. Previous studies suggest that the human endogenous retrovirus W family envelope (ERVWE1) is an influential risk factor for schizophrenia and inversely correlates with 5-HT4 receptor in schizophrenia. To our knowledge, no data describes the effect of ERVWE1 on 5-HT neuronal plasticity. N6-methyladenosine (m6A) regulates gene expression and impacts synaptic plasticity. Our research aims to systematically investigate the effects of ERVWE1 on 5-HT neuronal plasticity through m6A modification in schizophrenia.ResultsHTR1B, ALKBH5, and Arc exhibited higher levels in individuals with first-episode schizophrenia compared to the controls and showed a strong positive correlation with ERVWE1. Interestingly, HTR1B was also correlated with ALKBH5 and Arc. Further analyses confirmed that ALKBH5 may be an independent risk factor for schizophrenia. In vitro studies, we discovered that ERVWE1 enhanced HTR1B expression, thereby activating the ERK-ELK1-Arc pathway and reducing the complexity and spine density of 5-HT neurons. Furthermore, ERVWE1 reduced m6A levels through ALKBH5 demethylation. ERVWE1 induced HTR1B upregulation by improving its mRNA stability in ALKBH5-m6A-dependent epigenetic mechanisms. Importantly, ALKBH5 mediated the observed alterations in 5-HT neuronal plasticity induced by ERVWE1.ConclusionsOverall, HTR1B, Arc, and ALKBH5 levels were increased in schizophrenia and positively associated with ERVWE1. Moreover, ALKBH5 was a novel risk gene for schizophrenia. ERVWE1 impaired 5-HT neuronal plasticity in ALKBH5-m6A dependent mechanism by the HTR1B-ERK-ELK1-Arc pathway, which may be an important contributor to aberrant synaptic plasticity in schizophrenia.

Progressive Exaptation of Endogenous Retroviruses in Placental Evolution in Cattle.

Viviparity is made possible by the placenta, a structure acquired relatively recently in the evolutionary history of eutherian mammals. Compared to oviparity, it increases the survival rate of the fetus, owing to the eutherian placenta. Questions such as "How was the placenta acquired?" and "Why is there diversity in placental morphology among mammalian species?" remain largely unsolved. Our present understanding of the molecules regulating placental development remains unclear, owing in no small part to the persistent obscurity surrounding the molecular mechanisms underlying placental acquisition. Numerous genes associated with the development of eutherian placental morphology likely evolved to function at the fetal-maternal interface in conjunction with those participating in embryogenesis. Therefore, identifying these genes, how they were acquired, and how they came to be expressed specifically at the fetal-maternal interface will shed light on some crucial molecular mechanisms underlying placental evolution. Exhaustive studies support the hypothesis that endogenous retroviruses (ERVs) could be evolutional driving forces for trophoblast cell fusion and placental structure in mammalian placentas including those of the bovine species. This review focuses on bovine ERVs (BERVs) and their expression and function in the placenta.

Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus.

The PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, i.e. transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species' piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The unistrand cluster flamenco (flam) is essential in the somatic compartment of the Drosophila ovary to restrict Gypsy-family transposons from infecting the neighbouring germ cells. Disruption of flam results in transposon de-repression and sterility, yet it remains unknown whether this silencing mechanism is present more widely. Here, we systematically characterise 119 Drosophila species and identify five additional flam-like clusters separated by up to 45 million years of evolution. Small RNA-sequencing validated these as bona-fide unistrand piRNA clusters expressed in somatic cells of the ovary, where they selectively target transposons of the Gypsy family. Together, our study provides compelling evidence of a widely conserved transposon silencing mechanism that co-evolved with virus-like Gypsy-family transposons.

Cell identification, characterization, and documentation for use in the production of biological products

There is always a concern about the quality of cell-based products in terms of the contamination of the cells and their lack of efficiency. Therefore, it is very important to ensure these cells' identity, purity, efficacy, and suitability for the production of biological products and diagnostic uses. So, cells must be identified, evaluated, documented, and stored to be used consistently and efficiently. With these conditions, vaccine manufacturers have a suitable reserve of efficient and valuable cells for the production and quality control of biological products. In this review article, a strategic plan for cell-substrate well-characterization and identification according to scientific principles, the author's work experience, and regulatory guidance for the optimal use of that cell in research and diagnostic studies especially for the biological product production process have been drawn. For this purpose, all aspects of cell identification, cell evaluation, and cell characterization are discussed. Because of the importance of cell identity in the competence of a cell substrate, in the cell identification section, all aspects of cell identification, including general cell information, and specific cell characteristics, especially in terms of cell passage history, cell storage conditions, and cell coding and labeling were studied. In the part of cell evaluation and determination of cell characteristics, all tests necessary to determine cell characteristics from various aspects including determination of cell identity, cell growth conditions, cell quality, and efficiency, and the possibility of cell contamination with adventitious agents, including cellular, viral, bacterial, mycoplasma, and mycobacterial agents were introduced. Due to the importance of endogenous virus contamination, this topic is specially discussed. Also, the stability of the cell both from the aspect of genetic stability and from the aspect of stability of cell efficiency, were discussed. In the end, while reviewing the necessary documents to be under the control of the cell for use in the laboratory, based on the studies conducted, the certificate of the cell has been compiled. Therefore, on this basis, the studied cell can be used for research and diagnostic studies of virology, and especially for the production and quality control of biological products.

CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors.

Leukemic Cell-Intrinsic Innate Nucleic Acid Receptor Signaling Governs Efficacy of Hypomethylating Agents and Immunosurveillance in AML

Introduction: Hypomethylating agents (HMA), such as Azaciditine (AZA), are a long-established treatment option for acute myeloid leukemia (AML). Besides epigenetic reprogramming of malignant cell clones, HMAs are recognized to facilitate antineoplastic immunity. In vitro assessments in solid tumor models suggest that a major mechanism underlying AZA-triggered immune responses may be the activation of innate nucleic acid sensing pathways. As such, the RNA- and DNA-sensing receptor systems, RIG-I/MDA-5 and cGAS/STING - and subsequent type I interferon (IFN-I) signaling seem to be triggered by the transcriptional upregulation of otherwise DNA-hypermethylated and thus silenced endogenous retroviruses (ERVs). In AML, it is unknown whether similar processes are involved and/or by which molecular mechanisms, HMAs modulate anti-leukemic immunity against AML blasts, and how these processes are affected during acquired HMA resistance. Methods: Murine (c1498 and Wehi-3B) and human (MOLM-13) AML cell lines were exposed to AZA in vitro and analyzed for expression of ERVs. Nucleic acid receptor sensing pathways and IFN-I genes are not usually DNA methylated by AZA at promoter regions. However, the re-expression of endogenous nucleic acids or ERVs could trigger their activation. Protein and transcriptional expression of major components of nucleic acid receptor sensing pathways and IFN-I genes were, therefore, analysed. To understand the importance of AML cell-intrinsic nucleic acid receptor signaling in HMA-induced immunosurveillance, syngeneic c1498 and Wehi-3B AML models were employed. Mice received 2 cycles of 2.5mg/kg AZA (6 days on, 5 days off, 6 days on) intraperitoneally to mimic the principal treatment course used in patients. To address the contribution of the nucleic acid-sensing pathways to AZA treatment efficacy in vitroand in vivo, AML cells with acquired AZA resistance (AZA-R) or gene engineered AML cells deficient in RNA receptors (RIG-I/MDA-5), or the RNA- and DNA-sensing adapter proteins (MAVS and STING) were employed. Results: A targeted ERV screen revealed upregulation of various ERV transcripts in c1498 and Wehi-3B cells and was similarly observed in MOLM-13 AML cells following AZA exposure in vitro. Increased RNA transcript and protein expression of RIG-I/MAVS and (to a lesser degree) STING was observed, resulting in activation of downstream constituents including higher phosphorylation of the transcription factors IRF3/7 and NF-kB, IFN-I, and other ISGs. This was abrogated in RIG-I and MAVS, but not STING, deficient murine and human AML cells. Interestingly, expression of RIG-I/MDA-5, STING and all other downstream constituents, but not MAVS, were diminished in AZA-R AML cells. In mice harboring MAVS deficient AML (c1498), HMA treatment failed to show anti-leukemic activity resulting in rapid disease progress and death of recipient animals. Loss of STING in leukemic cells also impacted AZA treatment efficacy, although to a much less pronounced degree. These findings were similarly reproduced with Wehi-3B AML-engrafted mice. Following antibody-mediated depletion of T cells but not NK cells, mice rapidly succumbed to AML disease and demonstrated no anti-leukemic effect of AZA treatment. CyTOF and flow cytometric analysis also revealed increased bone marrow and spleen infiltration as well as activation of CD4 +/CD8 + T cells, NK cells, and MHC-II + APCs/Myeloid cells in AZA treated mice bearing wildtype AML, compared to untreated. This was not observed in AZA-treated mice bearing MAVS-deficient or AZA-R AML. Conclusion: Our findings indicate that nucleic acid receptor signaling (specifically RIG-I/MAVS) within leukemic cells is imperative for AZA efficacy and immunosurveillance in AML. Whether these RNA-sensing receptors are triggered by ERVs or alternate (endogenous) RNAs remains to be determined. Elucidating the molecular mechanisms of how nucleic acid sensors within leukemic blasts shape treatment efficacy of AZA may guide development of systematic immunotherapeutic strategies for AZA-R AML by targeted combinatorial approaches.

TET2-Mediated Dysregulation of Heterochromatin in Age-Related Clonal Hematopoiesis

Aging is one of the major negative factors of normal hematopoiesis, characterized by a decline in the self-renewal capabilities of hematopoietic stem and progenitor cells (HSPCs), alongside a myeloid lineage bias, thereby sabotaging the immune system and increasing the risk of malignant transformation. Epigenetic deregulation is a hallmark feature representative of aging and significantly contributes to age-related dysfunction in multiple systems. DNA methylation drifting is commonly observed during aging, especially within the partially methylated domains (PMDs). PMDs are intriguing heterochromatin regions featured by their late-replicating, lamina-associated, and B-compartment localized properties in HiC analysis. While heterochromatin is generally considered devoid of genes, it nonetheless plays a pivotal role in maintaining genomic stability. However, the link between age-related DNA methylation loss in PMDs and heterochromatin dysfunction remains elusive. In this study, we juxtapose the influence of aging on both wild-type (WT) and Tet2 knockout (KO) HSPCs. Tet2 depletion was found to counteract the functional deterioration typically associated with aging by preserving robust self-renewal and repopulation capacities in aging HSPCs. This resistance to functional decay may account for the age-related clonal hematopoiesis observed in Tet2KO HSPCs. At a molecular level, we identified distinct epigenetic regulatory mechanisms mediated by Dnmt3a and Tet2 at heterochromatin. These mechanisms are associated with alterations in 3D genome architecture during HSPC aging. Age-related dysregulation of heterochromatin leads to the upregulation of endogenous retroviruses (ERVs), which subsequently activate intracellular innate immune response and contribute to the functional decline of aging HSPCs. The application of reverse transcriptase inhibitors was shown to suppress ERV production and interferon-stimulated genes (ISGs) expression, thereby ameliorating age-related defects in aged HSPCs. Our findings provide compelling evidence supporting the intricate interplay between DNMT and TET in regulating DNA (de)methylation equilibrium in heterochromatin and euchromatin during stem cell aging. Moreover, our study offers critical insights into the mechanisms underlying age-related heterochromatin dysfunction, which contributes to the functional deterioration of HSPCs during aging.

Exploratory Analyses of Lymph Node Tissue, Serum, and Peripheral Blood Mononuclear Cells Do Not Reveal Pathogen Signatures in Idiopathic Multicentric Castleman Disease

Background : Castleman disease (CD) encompasses a group of hematologic disorders that share characteristic histopathological features. Unicentric CD (UCD) involves a solitary enlarged lymph node with typically mild symptoms. Multicentric CD (MCD) involves generalized lymphadenopathy and often severe cytokine-driven multi-organ dysfunction. MCD is further subdivided into cases caused by Human Herpesvirus-8 (HHV-8-associated MCD) infection, POEMS syndrome (POEMS-associated MCD), and those with an idiopathic cause (iMCD). The underlying pathological mechanisms and etiologies of UCD and iMCD are not well understood. Given that a viral pathogen, HHV-8, causes HHV-8-associated MCD, we pursued multiple approaches to identify potential pathogens that may trigger iMCD and UCD. Methods: We used 2 distinct detection methods to identify potential pathogens in CD. PathoChIP is a microarray-based assay that uses ~60,000 DNA/RNA probes to detect 4,000+ pathogens associated with human diseases. Viral Track is a computational method that can be applied to unmapped next-generation sequencing (NGS) reads to search for the presence of viral RNA. Formalin-fixed paraffin-embedded (FFPE) lymph node tissue was analyzed by PathoChIP from iMCD (n=6), UCD (n=8), HHV8-associated MCD (n=1, HHV8-MCD), EBV-driven post-transplant lymphoproliferative disorder (n=1, EBV-PTLD), reactive samples without a clear etiology (n=1), and POEMS-associated MCD (n=2) patients. We also analyzed serum from iMCD patients (n=2) and healthy donors (n=8). A ‘positive’ probe for a given microorganism was defined as exceeding the 95 th percentile on a per-probe basis of the optical signal captured across all probes in the dataset. Data from single-cell RNA sequencing of cryopreserved peripheral blood mononuclear cells (iMCD, n=4; EBV, n=2; HHV8, n=1; healthy donor, n=2) and bulk RNA sequencing of FFPE lymph node tissue (iMCD, n=9; sentinel, n=7; systemic lupus erythematosus (SLE), n=3; Diffuse large B-cell lymphoma (DLBCL), n=3) were used as input into the Viral Track program. To detect a positive viral signal, standard thresholds were required: >50 uniquely mapped reads to a given virus, a read complexity threshold >1.2, and >10% coverage of the length of the respective viral genome. Results: After quality control, 49,657 probes from the PathoCHIP assay were mapped to 4,000+ known pathogens. As expected, the HHV-8 MCD and EBV-PTLD samples identified significantly more positive probes compared to the rest of the cohort for HHV-8 (65/259 vs 1/259) and EBV (38/235 vs 1/235), respectively. Importantly, no other microorganisms were detected in UCD or MCD lymph nodes or serum samples at a significantly increased rate than controls. For Viral Track, human endogenous retrovirus K113 and Escherichia phages were detected at low levels throughout all results. Excluding these viruses, positive controls including HHV-8-MCD, and EBV solely identified their respective predicted virally mapped NGS reads and no viral signature was detected in healthy donors. Importantly, there was no significant viral signature specific to CD in PBMCs or LN tissue. Interestingly, 1 iMCD sample did contain a significant number of NGS sequencing reads mapping to HHV5. These results were confirmed with clinical data reporting a positive IgG, negative PCR, and negative IgM test within 2 weeks of the biopsy date, suggesting that the HHV5 positive result was likely detecting a previous, now latent infection rather than a potential pathological driver of CD. Conclusions: Although there are limitations to this study, including small cohorts and viral mRNA single-cell capture limitations, we were able to use 2 orthogonal pathogen detection methods on multiple tissue types from iMCD patients. Taken together, our results suggest that an active infectious agent is unlikely to be a major contributor or pathological mechanism driving UCD or iMCD. The continued search for potential causative microorganisms in iMCD patients should continue, but our results suggest that alternative hypotheses related to the pathology and etiology of iMCD and UCD should be prioritized.

Single-cell transcriptomics of human traumatic brain injury reveals activation of endogenous retroviruses in oligodendroglia

Traumatic brain injury (TBI) is a leading cause of chronic brain impairment and results in a robust, but poorly understood, neuroinflammatory response that contributes to the long-term pathology. We used single-nuclei RNA sequencing (snRNA-seq) to study transcriptomic changes in different cell populations in human brain tissue obtained acutely after severe, life-threatening TBI. This revealed a unique transcriptional response in oligodendrocyte precursors and mature oligodendrocytes, including the activation of a robust innate immune response, indicating an important role for oligodendroglia in the initiation of neuroinflammation. The activation of an innate immune response correlated with transcriptional upregulation of endogenous retroviruses in oligodendroglia. This observation was causally linked invitro using human glial progenitors, implicating these ancient viral sequences in human neuroinflammation. In summary, this work provides insight into the initiating events of the neuroinflammatory response in TBI, which has therapeutic implications.

Negative association between multiple sclerosis immunogenetic profile and in silico immunogenicities of 12 viruses

Human Leukocyte Antigen (HLA) is involved in both multiple sclerosis (MS) and immune response to viruses. Here we investigated the virus-HLA immunogenicity (V-HLA) of 12 viruses implicated in MS with respect to 17 HLA Class I alleles positively associated to MS prevalence in 14 European countries. Overall, higher V-HLA immunogenicity was associated with smaller MS-HLA effect, with human herpes virus 3 (HHV3), JC human polyoma virus (JCV), HHV1, HHV4, HHV7, HHV5 showing the strongest association, followed by HHV8, HHV6A, and HHV6B (moderate association), and human endogenous retrovirus (HERV-W), HHV2, and human papilloma virus (HPV) (weakest association). These findings suggest that viruses with proteins of high HLA immunogenicity are eliminated more effectively and, consequently, less likely to be involved in MS.

Shear stress in the intervillous space promotes syncytial formation of iPS cells-derived trophoblasts†.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72h of exposure to 10dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.

High-throughput complement component 4 genomic sequence analysis with C4Investigator.

The complement component 4 gene loci, composed of the C4A and C4B genes and located on chromosome 6, encodes for complement component 4 (C4) proteins, a key intermediate in the classical and lectin pathways of the complement system. The complement system is an important modulator of immune system activity and is also involved in the clearance of immune complexes and cellular debris. C4A and C4B gene loci exhibit copy number variation, with each composite gene varying between 0 and 5 copies per haplotype. C4A and C4B genes also vary in size depending on the presence of the human endogenous retrovirus (HERV) in intron 9, denoted by C4(L) for long-form and C4(S) for short-form, which affects expression and is found in both C4A and C4B. Additionally, human blood group antigens Rodgers and Chido are located on the C4 protein, with the Rodger epitope generally found on C4A protein, and the Chido epitope generally found on C4B protein. C4A and C4B copy number variation has been implicated in numerous autoimmune and pathogenic diseases. Despite the central role of C4 in immune function and regulation, high-throughput genomic sequence analysis of C4A and C4B variants has been impeded by the high degree of sequence similarity and complex genetic variation exhibited by these genes. To investigate C4 variation using genomic sequencing data, we have developed a novel bioinformatic pipeline for comprehensive, high-throughput characterization of human C4A and C4B sequences from short-read sequencing data, named C4Investigator. Using paired-end targeted or whole genome sequence data as input, C4Investigator determines the overall gene copy numbers, as well as C4A, C4B, C4(Rodger), C4(Ch), C4(L), and C4(S). Additionally, C4Ivestigator reports the full overall C4A and C4B aligned sequence, enabling nucleotide level analysis. To demonstrate the utility of this workflow we have analyzed C4A and C4B variation in the 1000 Genomes Project Data set, showing that these genes are highly poly-allelic with many variants that have the potential to impact C4 protein function.

HIV-1 Remission: Accelerating the Path to Permanent HIV-1 Silencing.

Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.

Does the interplay between human endogenous retrovirus K and extracellular vesicles contribute to aging?

The role of extracellular vesicles (EVs), including retroviral-like particles (RVLPs), in pathogenic processes is currently a subject of active investigation. Several studies have identified mechanistic links between the increased presence of EVs and the process of senescence. A recent study reveals that the reverse transcribed complementary DNA (cDNA) of a human endogenous retroviral sequence can activate the innate immune system and result in tissue damage and/or the spread of cellular senescence to distant tissues. Several studies have linked EVs to age-related diseases, such as Alzheimer's disease and Parkinson's disease, and have included isolation of EVs from individuals with these diseases. Loss of epigenetic regulation, immune activation, and environmental stimuli can all lead to the expression of endogenous retroviruses and the incorporation of their proteins and transcripts into EVs. In addition, EVs disseminating these endogenous retroviral components have now been shown to act in a paracrine manner in multiple human diseases. Further investigation of the connection between EVs containing endogenous retroviral protein products or nucleotides should be pursued in models of age-related diseases.

TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells.

DNA methylation deregulation at partially methylated domains (PMDs) represents an epigenetic signature of aging and cancer, yet the underlying molecular basis and resulting biological consequences remain unresolved. We report herein a mechanistic link between disrupted DNA methylation at PMDs and the spatial relocalization of H3K9me3-marked heterochromatin in aged hematopoietic stem and progenitor cells (HSPCs) or those with impaired DNA methylation. We uncover that TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses (ERVs) and interferon-stimulated genes (ISGs), hence contributing to functional decline of aged HSPCs. TET2-deficient HSPCs retain perinuclear distribution of heterochromatin and exhibit age-related clonal expansion. Reverse transcriptase inhibitors suppress ERVs and ISGs expression, thereby restoring age-related defects in aged HSPCs. Collectively, our findings deepen the understanding of the functional interplay between DNA methylation and histone modifications, which is vital for maintaining heterochromatin function and safeguarding genome stability in stem cells.

The Role of Human Endogenous Retrovirus (HERV)-K119 env in THP-1 Monocytic Cell Differentiation.

Human endogenous retrovirus (HERV)-K was reportedly inserted into the human genome millions of years ago and is closely related to various diseases, including cancer and immune regulation. In our previous studies, CRISPR-Cas9-enabled knockout (KO) of the HERV-K env gene was found to potentially reduce cell proliferation, cell migration, and invasion in colorectal and ovarian cancer cell lines. The immune response involves the migration and invasion of cells and is similar to cancer; however, in certain ways, it is completely unlike cancer. Therefore, we induced HERV-K119 env gene KO in THP-1, a monocytic cell that can be differentiated into a macrophage, to investigate the role of HERV-K119 env in immune regulation. Cell migration and invasion were noted to be significantly increased in HERV-K119 env KO THP-1 cells than in MOCK, and these results were contrary to those of cancer cells. To identify the underlying mechanism of HERV-K119 env KO in THP-1 cells, transcriptome analysis and cytokine array analysis were conducted. Semaphorin7A (SEMA7A), which induces the production of cytokines in macrophages and monocytic cells and plays an important role in immune effector cell activation during an inflammatory immune response, was significantly increased in HERV-K119 env KO THP-1 cells. We also found that HERV-K119 env KO THP-1 cells expressed various macrophage-specific surface markers, suggesting that KO of HERV-K119 env triggers the differentiation of THP-1 cells from monocytic cells into macrophages. In addition, analysis of the expression of M1 and M2 macrophage markers showed that M1 macrophage marker cluster of differentiation 32 (CD32) was significantly increased in HERV-K119 env KO cells. These results suggest that HERV-K119 env is implicated in the differentiation of monocytic cells into M1 macrophages and plays important roles in the immune response.