Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces.

Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces1-4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses5-7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines8,9 and the release of tissue-protective amphiregulin (AREG)10-12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions13-15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.

Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Joint single-cell measurements of nuclear proteins and RNA in vivo.

Identifying gene-regulatory targets of nuclear proteins in tissues is a challenge. Here we describe intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq), a scalable method that measures multiplexed intranuclear protein levels and the transcriptome in parallel across thousands of nuclei, enabling joint analysis of transcription factor (TF) levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed genome-wide associations of each TF and recovered known gene targets. TF-associated genes were coexpressed as distinct modules that each reflected positive or negative TF levels, showing that our approach can disentangle relative putative contributions of TFs to gene expression and add interpretability to inferred gene networks. inCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.

Spleen plays a major role in DLL4-driven acute T-cell lymphoblastic leukemia.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Peripheral elevation of TNF-α leads to early synaptic abnormalities in the mouse somatosensory cortex in experimental autoimmune encephalomyelitis.

Sensory abnormalities such as numbness and paresthesias are often the earliest symptoms in neuroinflammatory diseases including multiple sclerosis. The increased production of various cytokines occurs in the early stages of neuroinflammation and could have detrimental effects on the central nervous system, thereby contributing to sensory and cognitive deficits. However, it remains unknown whether and when elevation of cytokines causes changes in brain structure and function under inflammatory conditions. To address this question, we used a mouse model for experimental autoimmune encephalomyelitis (EAE) to examine the effect of inflammation and cytokine elevation on synaptic connections in the primary somatosensory cortex. Using in vivo two-photon microscopy, we found that the elimination and formation rates of dendritic spines and axonal boutons increased within 7 d of EAE induction--several days before the onset of paralysis--and continued to rise during the course of the disease. This synaptic instability occurred before T-cell infiltration and microglial activation in the central nervous system and was in conjunction with peripheral, but not central, production of TNF-α. Peripheral administration of a soluble TNF inhibitor prevented abnormal turnover of dendritic spines and axonal boutons in presymptomatic EAE mice. These findings indicate that peripheral production of TNF-α is a key mediator of synaptic instability in the primary somatosensory cortex and may contribute to sensory and cognitive deficits seen in autoimmune diseases.

Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-ß. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

Transcranial two-photon imaging of the living mouse brain.

This protocol describes imaging of the living mouse brain through a thinned skull using two-photon microscopy. This transcranial two-photon laser-scanning microscope (TPLSM) imaging method allows high-resolution imaging of fluorescently labeled neurons, microglia, astrocytes, and blood vessels, as well as subcellular structures such as dendritic spines and axonal varicosities. The surgical procedure that is required to allow imaging thins the cranium so that it becomes optically transparent. Once learned, the surgery can be performed in ∼30 min, and imaging can follow immediately. The procedure can be repeated multiple times, allowing brain cells and tissues to be studied in the same animals over short or long time intervals, depending on the design of the experiment. Two-photon imaging through a thinned and intact skull avoids side effects caused by skull removal and is a minimally invasive method for studying the living mouse brain under physiological and pathological conditions.

Target genes of the largest human SWI/SNF complex subunit control cell growth.

The largest subunit of the mammalian SWI/SNF-A or BAF (BRG1-associated factor) chromatin-remodelling complex is encoded by two related cDNAs hOsa1/BAF250a and hOsa2/BAF250b that are unique to the BAF complex and absent in the related PBAF (Polybromo BAF). hOsa/BAF250 has been shown to interact with transcriptional activators and bind to DNA suggesting that it acts to target the remodelling complex to chromatin. To better understand the functions of hOsa2, we established inducible stable HeLa cell lines over-expressing FLAG-hOsa2 or a derivative lacking the ARID (AT-rich interactive domain) DNA-binding domain. Immunopurification of complexes containing hOsa2 that was followed by mass spectrometry and immunoblotting demonstrated the presence of BRG1 and known BAFs, but not hOsa1 or hBRM. Deletion of the ARID did not compromise the integrity of the complex. Induction of hOsa2 expression caused impaired cell growth and accumulation of cells in the G0/G1 cell cycle phase. Elevated levels of the p53 and p21 proteins were detected in these cells while c-Myc mRNA and protein levels were found to decrease. Chromatin immunoprecipitation and reporter assays suggested that hOsa2 had a direct effect on c-myc and p21 promoter activity. Thus hOsa2 plays an important role in controlling genes regulating the cell cycle.

Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.

Imaging neurons, glia and vasculature in the living brain has become an important experimental tool for understanding how the brain works. Here we describe in detail a protocol for imaging cortical structures at high optical resolution through a thinned-skull cranial window in live mice using two-photon laser scanning microscopy (TPLSM). Surgery can be performed within 30-45 min and images can be acquired immediately thereafter. The procedure can be repeated multiple times allowing longitudinal imaging of the cortex over intervals ranging from days to years. Imaging through a thinned-skull cranial window avoids exposure of the meninges and the cortex, thus providing a minimally invasive approach for studying structural and functional changes of cells under normal and pathological conditions in the living brain.

Nuclear localization of the p75 neurotrophin receptor intracellular domain.

The p75 neurotrophin receptor, a member of the tumor necrosis factor superfamily of receptors, undergoes an alpha-secretase-mediated release of its extracellular domain, followed by a gamma-secretase-mediated intramembrane cleavage. Like amyloid precursor protein and Notch, gamma-secretase cleavage of the p75 receptor releases an intracellular domain (ICD). However, it has been experimentally challenging to determine the precise subcellular localization and functional consequences of the p75 ICD. Here, we utilized a nuclear translocation assay and biochemical fractionation approaches to follow the fate of the ICD. We found that the p75 ICD can translocate to the nucleus to activate a green fluorescent protein reporter gene. Furthermore, the p75 ICD was localized in nuclear fractions. Chromatin immunoprecipitation experiments indicated that nerve growth factor induced the association of endogenous p75 with the cyclin E(1) promoter. Expression of the p75 ICD resulted in modulation of gene expression from this locus. These results suggest that the p75 ICD generated by gamma-secretase cleavage is capable of modulating transcriptional events in the nucleus.

The role of YY1 in reduced HP1alpha gene expression in invasive human breast cancer cells.

INTRODUCTION: Heterochromatin protein 1 (HP1) associates with chromatin by binding to histone H3 and contributes to gene silencing. There are three isoforms of HP1 in mammals: HP1alpha, beta, and gamma. Studies have shown that the level of HP1alpha is reduced in invasive human breast cancer cell lines such as MDA-MB-231 and HS578T compared with non-invasive cell lines such as MCF7 and T47D. It is hypothesized that reduced HP1alpha expression may lead to impaired epigenetic silencing of genes that are important in the acquisition of an invasive phenotype. We set out to determine whether reduced expression of HP1alpha in invasive breast cancer cell lines occurs at the level of transcription. METHODS: We used transient transfection assays to investigate the mechanism of differential transcriptional activity of the human HP1alpha gene promoter in different cell lines. Mutational analysis of putative transcription factor binding sites in an HP1alpha gene reporter construct was performed to identify transcription factors responsible for the differential activity. SiRNA-mediated knockdown and chromatin immunoprecipitation experiments were performed to determine the role of a specific transcription factor in regulating the HP1alpha gene. RESULTS: The transcription factor yin yang 1 (YY1) was found to play a role in differential transcriptional activity of the HP1alpha gene. Examination of the YY1 protein and mRNA levels revealed that both were reduced in the invasive cell line HS578T compared with MCF7 cells. YY1 knockdown in MCF7 cells resulted in a decreased level of HP1alpha mRNA, indicating that YY1 positively regulates HP1alpha expression. Chromatin immunoprecipitation experiments verified YY1 occupancy at the HP1alpha gene promoter in MCF7 cells but not HS578T cells. Overexpression of YY1 in HS578T cells decreased cell migration in a manner independent of HP1alpha overexpression. CONCLUSIONS: Our data suggests that a reduction of YY1 expression in breast cancer cells could contribute to the acquisition of an invasive phenotype through increased cell migration as well as by reduced expression of HP1alpha.