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Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous neurodegenerative disorder that affects motor neurons in the brain and spinal cord, causing progressive loss of voluntary muscle control1,2. ALS heterogeneity includes the age of manifestation, the rate of progression, and the anatomical sites of symptom onset. In addition, disease-causing mutations in specific genes have been identified and are used to catalog different subtypes of ALS3. Interestingly, several ALS-associated genes have been shown to affect immune functions, and a variety of aberrant inflammatory events have been reported in patients and mouse models4-11, suggesting that specific immune features can also account for ALS heterogeneity. ALS4 is characterized by juvenile-onset and slow progression12. After experiencing mild symptoms during their childhood, ALS4 patients show motor difficulties by their 30s, and most of them require walkers or wheelchairs by their 50s. ALS4 is caused by dominant mutations in the gene SETX. Using Setx knock-in (KI) mice carrying the ALS4 causative L389S mutation, we discovered an immunological signature consisting of clonally activated CD8 T cells specifically in the central nervous system and blood of KI animals. Expansion of antigen-specific CD8 T cells mirrors disease progression. Bone marrow transplantation experiments indicate an essential role of the immune system in ALS4 neurodegeneration. Furthermore, we found that clonally expanded CD8 T cells circulate in the peripheral blood of ALS4 patients. Our results provide evidence of an antigen-specific CD8 T cell response linked to ALS4, and can serve not only to unravel specific disease mechanisms, but as a potential biomarker of disease activity.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans., Graphical abstract, Inhibition of topoisomerase 1 through the FDA-approved molecule topotecan suppresses SARS-CoV-2-infection-associated lethal inflammation in hamster and mouse models without compromising antiviral immune responses.

Acquired mutations in DNMT3A are common in blood, but their impacts on mature immune cells are poorly understood. Lim et al. demonstrate that DNMT3A defects cause dichotomous DNA methylation defects at enhancers, leading to altered enhancer activity and abnormal gene expression in mature immune cells., DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations., Graphical Abstract

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, as viral polymerases cleave 5’-m7G-capped host transcripts to primeviral mRNA synthesis (‘cap-snatching’). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, that we named ‘start-snatching’. Depending on the reading frame, start-snatching allows the translation of host and viral “untranslated regions” (UTRs) to create Nterminallyextended viral proteins or entirely novel polypeptides by geneticoverprinting. We show that both types of chimeric proteins are made in IAVinfectedcells, generate T cell responses and contribute to virulence. Our resultsindicate that during infection with IAV, and likely a multitude of other human-,animal- and plant-viruses, a host-dependent mechanism allows the genesis ofhybrid genes.

Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment.

Transcription factor (TF)‐based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial‐like precursors that generate hematopoietic stem progenitor (HSPC)‐like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC‐like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell‐based therapeutics., FEBS Letters, 593 (23), ISSN:0014-5793, ISSN:1873-3468

Motor neuron disorders (MND) cause disability and death globally. Despite an increasing knowledge of the genetic defects of MND, a clear understanding of the mechanisms of disease initiation and progression is lacking. Recent advance in the field has highlighted that several genes linked to MND are essential for maintaining immune homeostasis, suggesting that dysfunctions in the immune system could influence the development of the disease or aggravate its pathology. Amyotrophic Lateral Sclerosis 4 (ALS4) is a rare, juvenile form of MND. It is caused by mutations in the gene SENATAXIN (SETX), which encodes for a nuclear helicase ubiquitously expressed. Our group identified SETX as a transcriptional regulator of inflammatory responses. Since SETX is involved in both MND and the control of the immune response, we hypothesized that underlying defects in the immune system could be associated to ALS4 progression. We took advantage of the Setx-L389S+/− knock-in (KI) mouse carrying the most common causative variant of ALS4 in humans. Surprisingly, we found that SetxL389S needs to be expressed in both neuronal and immune cells to trigger disease. In parallel, we discovered a pathologic signature consisting of high frequencies of activated CD8 T cells in the central nervous system (CNS) and blood of KI animals. Further analyses showed clonal expansion of aberrant activated CD8 T cells. Dysfunctions in the CD8 T cell compartment have been confirmed in the peripheral blood of ALS4 patients, as compared to age-matched controls. Collectively, our results suggest that ALS4 is characterized by aberrant CD8 T cell responses of possible autoimmune origin. Our findings have the potential to pave the way for immune-based diagnosis and therapy for patients.