Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] . Blocking either viral replication with remdesivir 21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.Acute SARS-CoV-2 infection resolves in most patients but becomes chronic and sometimes deadly in about 10-20% of patients [1][2][3][4][5][6][7][14][15][16]20,[24][25][26][27] . Two hallmarks of severe COVID-19 are a sustained IFN response and viral RNA persisting for months [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]20,[24][25][26][27][28] . This chronicity is recapitulated in SARS-CoV-2-infected MISTRG6-hACE2 humanized mice 19 . High levels of IL-1β, IL-18 and lactate dehydrogenase (LDH) are correlated with COVID-19 severity in patients, suggesting a role for inflammasome activation and pyroptosis in pathology [5][6][7][14][15][16][17][18]29 . Here we show that human lung macrophages are infected by SARS-CoV-2. Replicating SARS-CoV-2 in these human macrophages activates inflammasomes and initiates an inflammatory cascade with a unique transcriptome, results in pyroptosis, and contributes to the downstream type-I IFN response. Blocking viral replication, the downstream IFN response or inflammasome activation in vivo during the chronic phase of the disease attenuates many aspects of the overactive immune inflammatory response (especially the inflammatory macrophage response) and disease. Viral replication and the IFN responseChronic interferon is associated with disease severity and impaired recovery in influenza infection 30 . To test whether a viral-RNAdependent type-I IFN response was a driver of chronic disease, we treated SARS-CoV-2-infected MISTRG6-hACE2 mice with remdesivir [21][22][23] and/or anti-IFNAR2 antibodies (Fig. 1a) to inhibit vi...

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts 1 . Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types 2 , 3 . However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans . By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2 -expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E 2 (PGE 2 ). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE 2 drives the expansion οf a population of Sca-1 + reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1 + cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE 2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1 + cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE 2 –Ptger4. Analyses of patient-derived organoids established that PGE 2 –PTGER4 also regulates stem cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2 -expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE 2 –Ptger4–Yap signalling axis.

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging their acquired effector function. Metabolic reprograming from the naïve-state is required throughout these phases of activation1. In CD4 T cells, T cell receptor (TCR) ligation, along with co-stimulatory and cytokine signals induce a glycolytic anabolic program required for biomass generation, rapid proliferation, and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodeling) and function are coordinately orchestrated by signal transduction and transcriptional remodeling; however, it remains unclear whether these processes are independently regulated by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support T helper 1 (Th1) cell differentiation and effector function, biochemically uncoupling these processes. We find that the TCA cycle is required for terminal Th1 cell effector function through succinate dehydrogenase (SDH; Complex II), yet the activity of SDH suppresses Th1 cell proliferation and histone acetylation. In contrast, we show that Complex I of the electron transport chain (ETC), the malate-aspartate shuttle, and citrate export from the mitochondria are required to maintain aspartate synthesis necessary for Th cell proliferation. Furthermore, we find that mitochondrial citrate export and malate-aspartate shuttle promote histone acetylation and specifically regulate the expression of a set of genes involved in T cell activation, proliferation, and metabolic programming. Combining genetic, pharmacological, and metabolomics approaches, we demonstrate that T helper cell differentiation and terminal effector function can be biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, citrate export, and Complex I supply the substrates needed for proliferation and epigenetic remodeling during early T cell activation, while Complex II consumes the substrates of these pathways, antagonizing differentiation and enforcing terminal effector function. Our data suggest that transcriptional programming works in concert with a parallel biochemical network to enforce cell state.

OVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 , is a heterogenous disease with few therapeutic options. Although anti-viral immunity mediates viral clearance in mild COVID-19, robust inflammatory cytokines, decreased circulating lymphocytes and dysregulated myeloid and lymphocyte compartments characterize immunopathology in severe ). Accurate model systems are essential to evaluate promising discoveries, but most available rodent and non-human primate models do not reveal the immunopathology seen in patients and are not well suited to test therapeutics in the context of severe COVID-19 or post-acute sequelae of ).ACE2 is a SARS-CoV-2 receptor [20][21][22] . SARS-CoV-2 does not infect standard laboratory mice owing to differences between mouse and human ACE2 (hACE2) that limit viral entry 14,15,[20][21][22] . Mice with transgenic or transient hACE2 expression can be infected with SARS-CoV-2 (refs. 14,[16][17][18][19] ). Although acute viral response, transmission and vaccine efficacy can be tested in these models, they lack severe, chronic disease (Extended Data Fig. 1). We hypothesized that a functional human immune system would model innate and adaptive human immunity during SARS-CoV-2 infection and confer chronicity and pathology seen in patients. Mice with a human immune system (humanized mice), generated via transplantation of human hematopoietic stem and progenitor cells (HSPCs), are invaluable tools to study the human immune system in vivo 23,24 . MISTRG6 (ref. 25 ) mice were engineered by a human/mouse homolog gene replacement strategy to provide physiological expression with regard to quantity, location and time of M-CSF (monocytes and tissue macrophage development) 26 , GM-CSF/IL3 (lung alveolar macrophages) 27 , SIRPα (macrophage tolerance to human cells) 28 , ThPO (hematopoiesis and platelets) 29 and IL6 (improved engraftment and antibody responses) 25,30,31 in a Rag2/γ common chain deleted background. When engrafted with human HSPCs, these mice have a comprehensive immune system similar to humans 25,32 . Delivering hACE2 via an adeno-associated virus (AAV) vector to the lungs allows SARS-CoV-2 infection of HSPC-engrafted MISTRG6 mice. Thus, we created a humanized mouse model of COVID-19 that recapitulates the human innate and adaptive immune systems that is amenable to the mechanistic study of COVID-19 and its myriad of complications.

Highlights d Epithelial and immune cell IL-18 are not required to combat S. typhimurium d Enteric neurons express IL-18 d Enteric neuronal IL-18 controls goblet cell antimicrobial protein expression d Neuronal IL-18 directs killing of enteric bacterial pathogens