Metabolites derived from the intestinal microbiota, including bile acids (BA), extensively modulate vertebrate physiology, including development1, metabolism2, 3-4, immune responses5, 6-7 and cognitive function8. However, to what extent host responses balance the physiological effects of microbiota-derived metabolites remains unclear9,10. Here, using untargeted metabolomics of mouse tissues, we identified a family of BA-methylcysteamine (BA-MCY) conjugates that are abundant in the intestine and dependent on vanin 1 (VNN1), a pantetheinase highly expressed in intestinal tissues. This host-dependent MCY conjugation inverts BA function in the hepatobiliary system. Whereas microbiota-derived free BAs function as agonists of the farnesoid X receptor (FXR) and negatively regulate BA production, BA-MCYs act as potent antagonists of FXR and promote expression of BA biosynthesis genes in vivo. Supplementation with stable-isotope-labelled BA-MCY increased BA production in an FXR-dependent manner, and BA-MCY supplementation in a mouse model of hypercholesteraemia decreased lipid accumulation in the liver, consistent with BA-MCYs acting as intestinal FXR antagonists. The levels of BA-MCY were reduced in microbiota-deficient mice and restored by transplantation of human faecal microbiota. Dietary intervention with inulin fibre further increased levels of both free BAs and BA-MCY levels, indicating that BA-MCY production by the host is regulated by levels of microbiota-derived free BAs. We further show that diverse BA-MCYs are also present in human serum. Together, our results indicate that BA-MCY conjugation by the host balances host-dependent and microbiota-dependent metabolic pathways that regulate FXR-dependent physiology.

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.

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.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment1–7. An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system’s (CNS) resident professional phagocyte8–10. Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo. Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

The appropriate development of macrophages, the body's professional phagocyte, is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity.Highlights:Myeloid-specific WNK1 loss causes failed macrophage development and premature deathM-CSF-stimulated myeloid progenitors and precursors become neutrophils instead of macrophagesM-CSF induces macropinocytosis by myeloid progenitors, which depends on WNK1Macropinocytosis enforces macrophage lineage commitment.

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.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites(1,2), although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)-induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

Sepsis is a life-threatening systemic inflammatory condition causing approximately 11 million annual deaths worldwide. Although key hyperinflammation-based organ dysfunctions that drive disease pathology have been recognized, our understanding of the factors that predispose patients to septic mortality is limited. Due to the lack of reliable prognostic measures, the development of appropriate clinical management that improves patient survival remains challenging. Here, we discovered that a subpopulation of CD49chigh neutrophils with dramatic upregulation of the complement component 1q (C1q) gene expression arises during severe sepsis. We further found that deceased septic patients failed to maintain C1q protein expression in their neutrophils, whereas septic survivors expressed higher levels of C1q. In mouse sepsis models, blocking C1q with neutralizing antibodies or conditionally knocking out C1q in neutrophils led to a significant increase in septic mortality. Apoptotic neutrophils release C1q to control their own clearance in critically injured organs during sepsis; thus, treatment of septic mice with C1q drastically increased survival. These results suggest that neutrophil C1q is a reliable prognostic biomarker of septic mortality and a potential novel therapeutic target for the treatment of sepsis.

Severe coronavirus disease 2019 (COVID-19) is characterized by systemic inflammation and can result in protracted symptoms. Robust systemic inflammation may trigger persistent changes in hematopoietic cells and innate immune memory through epigenetic mechanisms. We reveal that rare circulating hematopoietic stem and progenitor cells (HSPC), enriched from human blood, match the diversity of HSPC in bone marrow, enabling investigation of hematopoiesis and HSPC epigenomics. Following COVID-19, HSPC retain epigenomic alterations that are conveyed, through differentiation, to progeny innate immune cells. Epigenomic changes vary with disease severity, persist for months to a year, and are associated with increased myeloid cell differentiation and inflammatory or antiviral programs. Epigenetic reprogramming of HSPC may underly altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors. One Sentence Summary Transcriptomic and epigenomic analysis of blood reveal sustained changes in hematopoiesis and innate immunity after COVID-19. Graphical Abstract

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 surfaces 1 – 4 . ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses 5 – 7 . However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines 8 , 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 functions 13 – 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.

Rationale: The coronavirus disease (COVID-19) pandemic has led to a dramatic increase in the number of survivors of critical illness. These survivors are at increased risk for physical, psychological, and cognitive impairments known collectively as post-intensive care syndrome (PICS). Little is known about the prevalence of PICS in COVID-19 survivors. Objectives: To report the prevalence of physical, psychological, and cognitive impairment among COVID-19 intensive care unit (ICU) survivors receiving follow-up care in an ICU recovery clinic, to assess for associations between PICS and ICU-related factors, and to compare the cohort of ICU survivors who attended a post-ICU clinic with a cohort of ICU survivors who did not. Methods: We performed a retrospective cohort study of COVID-19 ICU survivors admitted from March to May 2020 who were subsequently seen in a post-ICU recovery clinic in New York City. We abstracted medical chart data on available clinical screening instruments for physical, psychological, and cognitive impairment. Associations between these outcomes and care-related variables were tested. Baseline characteristics and in-hospital treatments of the post-ICU clinic cohort were compared with those of COVID-19 ICU survivors from the same institution who were not seen in the post-ICU clinic. Results: Eighty-seven COVID-19 ICU survivors were seen in our post-ICU recovery clinic. The median age was 62 years, and 74% were male. The median length of hospitalization was 51 days, and the median length of ICU stay was 22 days. At the post-ICU follow-up visit, 29%, 21%, and 13% of patients reported clinically significant levels of depressive symptoms, anxiety, and post-traumatic stress disorder symptoms, respectively. Twenty-five percent had cognitive impairment. The overall prevalence of PICS was 90%. There were no associations between length of ICU stay, delirium, and exposure to benzodiazepines, steroids, or systemic paralytics with positive screening results for physical, psychological, or cognitive impairment. Baseline characteristics and ICU-related factors were similar in the cohort of COVID-19 ICU survivors who attended the ICU recovery clinic and those who did not. Conclusions: PICS is common in COVID-19 survivors. We did not find any association with length of ICU stay or the use of benzodiazepines, steroids, or paralytics.

ABSTRACTVascular injury is a menacing element of acute respiratory distress syndrome (ARDS) pathogenesis. To better understand the role of vascular injury in COVID-19 ARDS, we used lung autopsy immunohistochemistry and blood proteomics from COVID-19 subjects at distinct timepoints in disease pathogenesis, including a hospitalized cohort at risk of ARDS development (“at risk”, N=59), an intensive care unit cohort with ARDS (“ARDS”, N=31), and a cohort recovering from ARDS (“recovery”, N=12). COVID-19 ARDS lung autopsy tissue revealed an association between vascular injury and platelet-rich microthrombi. This link guided the derivation of a protein signature in the at risk cohort characterized by lower expression of vascular proteins in subjects who died, an early signal of vascular limitation termed the maladaptive vascular response. These findings were replicated in COVID-19 ARDS subjects, as well as when bacterial and influenza ARDS patients (N=29) were considered, hinting at a common final pathway of vascular injury that is more disease (ARDS) then cause (COVID-19) specific, and may be related to vascular cell death. Among recovery subjects, our vascular signature identified patients with good functional recovery one year later. This vascular injury signature could be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

A 68-year-old man with a history of recent COVID-19 ARDS with prolonged respiratory failure requiring a tracheostomy was transferred to the medical ICU because of new-onset shock. The patient's hospital course from COVID-19 was complicated by ventilator-associated pneumonia attributable to Pseudomonas aeruginosa and Klebsiella pneumoniae, which were successfully treated, and a left femoral vein DVT requiring therapeutic anti-coagulation with enoxaparin. On admission to the ICU, the patient was hypotensive, requiring norepinephrine at 28 μg/min, and phenylephrine at 100 μg/min to maintain a systolic BP of 90 mm Hg. Bedside physical examination was notable for lethargy, and laboratory results were relevant for a lactate of 8 mmol/L and a hemoglobin of 5.6 g/dL, which was a decrease from 9.7 g/dL 24 hours earlier. A portable chest radiograph illustrated reticular opacities at the bases (shown in Fig 1). A focused bedside ultrasound assessment of the heart, lungs, and abdomen was performed. Relevant findings are illustrated in Videos 1 and 2, which were obtained using a phased array transducer oriented in the coronal plane in the patient's right 4th and 6th intercostal spaces at the midaxillary line. Video 3 is taken at the 6th intercostal space posterior axillary line. All videos were obtained while the patient was in the sitting position at 60 degrees. Question: What do the ultrasound findings in Video 1 illustrate, and what is the most likely cause of the patient's shock? Answer: The series of videos show an extra thoracic fluid collection outside of the chest wall with an anechoic region representing blood and a hyperechoic area consistent with a hematoma suggesting hemorrhage as the etiology of shock Video 1 obtained from the 4th intercostal space at the midaxillary line shows that the lung, diaphragm, chest wall, and liver are all continuous; therefore, the anechoic structure that is present is located outside the thorax and within the soft tissue of the chest. Video 2 is taken from the 6th intercostal space, along the same line as Video 1. Video 2 provides illustration of the key anatomical relationship that exists between the rib spaces and the pleura, as the visualized pleural line on ultrasound is 0.5 cm below the periosteal reflection of the ribs regardless of habitus.1Cardenas-Garcia J. Mayo P.H. Folch E. Ultrasonographic evaluation of the pleura.2015: 2https://doi.org/10.1177/2373997515610270Crossref Google Scholar Identifying this key relationship confirms that the anechoic collection with fibrous stranding and the hematoma that is visualized is located outside of the thorax. Video 3, taken from the posterior axillary line along the 6th intercostal space, once again confirms that the fluid collection is outside the chest, as the juxtaposition of the liver, lung, and chest wall can be appreciated (Please see Discussion Video for further explanation). Given the new-onset shock, decrease in hemoglobin, therapeutic dosing of anticoagulation, and ultrasound findings of a complex fluid collection outside the chest, hemorrhagic shock from a spontaneous intercostal arterial bleed became the leading diagnosis. The blood supply of the intercostal spaces within the chest arises from the intercostal arteries, which have anterior and posterior components. The anterior intercostal arteries, if coming from the first six intercostal spaces, are fed by the internal thoracic artery, and below the 6th intercostal space by the musculophrenic artery. The first two posterior intercostal arteries come from the costocervical trunk; from the 3rd rib space downward the posterior intercostal artery arises directly from the aorta.2Brennan P.A. Standring S. Wiseman S.M. Gray's Surgical Anatomy. Elsevier, Amsterdam2020Google Scholar The blood flow within the intercostal arteries is quite robust, with flow reaching up to 70 mL/min on the right and 99.5 mL/min on the left, highlighting how a patient can quickly develop shock if these vessels were to bleed.3Koyanagi T. Kawaharada N. Kurimoto Y. et al.Examination of intercostal arteries with transthoracic Doppler sonography.Echocardiography. 2010; 27: 17-20Crossref PubMed Scopus (22) Google Scholar The overall incidence of chest wall hematoma caused by intercostal arterial bleeding is unknown, but the few case reports available suggest that it is a rarely encountered condition, with causes being iatrogenesis, chest wall trauma, or spontaneous.4Roddy S.P. Transcatheter arterial embolization for the management of iatrogenic and Blunt TRAUMATIC intercostal artery injuries.J Vasc Surg. 2009; 49: 1624Abstract Full Text Full Text PDF Scopus (2) Google Scholar,5Jang J.Y. Lim Y.S. Woo J.H. Jang J.H. Spontaneous rupture of intercostal artery after severe cough.Am J Emerg Med. 2015; 33: e1-e3Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar When examining the chest with ultrasound, a hematoma can have a variety of appearances depending on the age of the lesion. In the acute stage, a hematoma appears as an echogenic structure, progressing to a hypoechoic structure over time, with septations observed in 44% of hematomas after 5 days of being present.6Wicks J. Silver T. Bree R. Gray scale features of hematomas: an ultrasonic spectrum.Am J Roentgenol. 1978; 131: 977-980Crossref PubMed Scopus (90) Google Scholar Enoxaparin is a low-molecular-weight heparin (LMWH) that is commonly given for DVT and pulmonary embolus at a dosage of 1 mg/kg twice daily. In patients receiving enoxaparin for DVT or pulmonary embolus, the incidence of major bleeding (defined as being clinically overt and associated with a hemoglobin decrease of 2 g/dL or requiring a transfusion of 2 units of packed RBCs) is 2.9% at 3 months.7Mismetti P. Quenet S. Levine M. et al.Enoxaparin in the treatment of deep vein thrombosis with or without pulmonary embolism.Chest. 2005; 128: 2203-2210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Monitoring of antithrombotic effects is not usually necessary but can be considered in obese patients and those with renal dysfunction, targeting an anti-Xa level of 0.6 to 1.0 units/mL measured 4 hours after the third dose. In patients receiving LMWH, such as enoxaparin, there is no proven reversal agent. Protamine can neutralize the anti-IIa activity of LMWH; however, the effect of neutralization on anti-Xa is variable. Nonetheless, CHEST recommends that protamine be given at a dosage of 1 mg per 100 anti-Xa units, up to a maximum single dose of 50 mg.8Hirsh J. Bauer K.A. Donati M.B. Gould M. Samama M.M. Weitz J.I. Parenteral anticoagulants.Chest. 2008; 133: 141S-159SAbstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar The patient was resuscitated with two units of packed RBCs, and a CT of the chest with IV contrast was performed, which showed a large right posterolateral chest wall hematoma with active arterial extravasation from the 4th and 5th intercostal arteries, confirming the diagnosis of a spontaneous intercostal arterial bleed. Interventional radiology was consulted, and the patient underwent successful embolization, with resolution of his shock state shortly thereafter. 1.In patients with undifferentiated shock in the ICU, bedside assessment with point-of-care ultrasound can be valuable in narrowing the differential.2.Identification of the periosteal reflection from the ribs aids in visualization of the pleural line, which is located 0.5 cm below the reflection. Recognition of this anatomic relationship on chest ultrasound can help identify whether the abnormality is intrathoracic or extrathoracic.3.Hematomas have a variable sonographic presentation, appearing more echogenic early on and becoming hypoechoic over time. Financial/nonfinancial disclosures: None declared. Other contributions: CHEST worked with the authors to ensure that the Journal policies on patient consent to report information were met. Additional information: To analyze this case with the videos, see the online article. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3ZWEyMmQ2YjViMjUxNzg1MTk2M2ZmOTAzZDk4NjIwMiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNzEzNDgxOTEzfQ.ISpnEnOK6-b2qG4jp4_bHh22rVJ2sDuspPJqkj2MWrOJtpQVn0q0_ktW19-g64W920W6I8J_g7rCFh1zOgJg9mcsAVFrvQWmgtdA6ZCuyzfGgQiE4W-b3KJ8hsJNITJ8YaD7OpwijWM3eY10Tmy0aBQOYnsb0ESA8CumevEaFUpaisNlefiatAuhSvihws86gUsPe8Xo_ao7LIcb0zTi8_QiAoLMSgEgJxQkd7iZE_n7juZCGT7VHqYwog07G170CPFqNfas6w0rmobDv6sReWjw15XIKQZtbR69fCBx8-rDe7EjerhElvoaJBYucqUcMtwUHHgXsYCkLgc-9E6lDw Download .mp4 (12.09 MB) Help with .mp4 files Video 1eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3NDlmMjYwNDc5ZTA1ODcyY2Q1ZTk5YmI3MGUyNTk4YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNzEzNDgxOTE0fQ.iEaO8mCa1inUqLw2lNOrq8OBuBiKHTQ_KOeBxkH-tkClaf3xBPQWBpPtKGgru8mal1skkeMUemBeGYkHZGWIGswRxCnmPvhNb9ZoXcPYl8YbgR2mygImhCXPWGwJWg3Fa1g9nsu7wT8WXwr7atSMXs2gcezOSicm223xYL9EaYjj_8kypDg90kqaHnCNHwdwzH0K9kXBkdgcrSoNEYlJ3tey9dim92-HTC1Rg3-GdTPeN6YUKkxRkY_OEBD_BaLZxQCZvJsECAsEL178iUmsUFI-e-YYeY73oqWBdrITfquqr_dgVpf_mcJFOzMLwW4QXrCLy7LR97tmU6_ZAjm5bQ Download .mp4 (16.09 MB) Help with .mp4 files Video 2

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.

Identifying gene regulatory targets of nuclear proteins in tissues remains a challenge. Here we describe intranuclear Cellular Indexing of Transcriptomes and Epitopes (inCITE-seq), a scalable method for measuring multiplexed intranuclear protein levels and the transcriptome in parallel in thousands of cells, enabling joint analysis of 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 the genome-wide effect of each TF and recovered known targets. Cell type-specific genes associated with each TF were co-expressed as distinct modules that each corresponded to positive or negative TF levels, showing that our approach can disentangle relative contributions of TFs to gene expression and add interpretability to 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.

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases1–6, but the pathways that control these responses remain unclear. Here we define a population of inflammatory group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen-presenting cells that restimulate myelin-specific T cells, and are increased in individuals with multiple sclerosis. Notably, antigen presentation by inflammatory ILC3s is required to promote T cell responses in the CNS and the development of multiple-sclerosis-like disease in mouse models. By contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but instead limit autoimmune T cell responses and prevent multiple-sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define a population of inflammatory ILC3s that is essential for directly promoting T-cell-dependent neuroinflammation in the CNS and reveal the potential of harnessing peripheral tissue-resident ILC3s for the prevention of autoimmune disease. A subset of inflammatory group 3 innate lymphoid cells, here termed iILC3s, infiltrate the central nervous system and promote neuroinflammation and disease progression in a mouse model of multiple sclerosis.

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.

Group 2 innate lymphoid cells (ILC2s) reside in multiple tissues, including lymphoid organs and barrier surfaces, and secrete type 2 cytokines including interleukin-5 (IL-5), IL-9, and IL-13. These cells participate in multiple physiological processes including allergic inflammation, tissue repair, metabolic homeostasis, and host defense against helminth infections. Recent studies indicate that neurotransmitters and neuropeptides can play an important role in regulating ILC2 responses; however, the mechanisms that underlie these processes in vivo remain incompletely defined. Here, we identify that activated ILC2s up-regulate choline acetyltransferase (ChAT)-the enzyme responsible for the biosynthesis of acetylcholine (ACh)-after infection with the helminth parasite Nippostrongylus brasiliensis or treatment with alarmins or cytokines including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). ILC2s also express acetylcholine receptors (AChRs), and ACh administration promotes ILC2 cytokine production and elicits expulsion of helminth infection. In accordance with this, ChAT deficiency in ILC2s leads to defective ILC2 responses and impaired immunity against helminth infection. Together, these results reveal a previously unrecognized role of the ChAT-ACh pathway in promoting type 2 innate immunity to helminth infection.