Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring ...cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called "priming loop", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.
All coronaviruses known to have recently emerged as human pathogens probably originated in bats
. Here we use a single experimental platform based on immunodeficient mice implanted with human lung ...tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.
The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins ...inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.
Background & Aims We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we ...describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Methods We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori . Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses. Results Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response. Conclusions We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.
The intestinal epithelium is a highly dynamic structure that rejuvenates in response to acute stressors and can undergo alterations in cellular composition as animals age. The microbiota, acting via ...secreted factors related to indole, appear to regulate the sensitivity of the epithelium to stressors and promote epithelial repair via IL-22 and type I IFN signaling. As animals age, the cellular composition of the intestinal epithelium changes, resulting in a decreased proportion of goblet cells in the colon. We show that colonization of young or geriatric mice with bacteria that secrete indoles and various derivatives or administration of the indole derivative indole-3 aldehyde increases proliferation of epithelial cells and promotes goblet cell differentiation, reversing an effect of aging. To induce goblet cell differentiation, indole acts via the xenobiotic aryl hydrocarbon receptor to increase expression of the cytokine IL-10. However, the effects of indoles on goblet cells do not depend on type I IFN or on IL-22 signaling, pathways responsible for protection against acute stressors. Thus, indoles derived from the commensal microbiota regulate intestinal homeostasis, especially during aging, via mechanisms distinct from those used during responses to acute stressors. Indoles may have utility as an intervention to limit the decline of barrier integrity and the resulting systemic inflammation that occurs with aging.
The hallmark features of type 2 mucosal immunity include intestinal tuft and goblet cell expansion initiated by tuft cell activation. How infectious agents that induce type 2 mucosal immunity are ...detected by tuft cells is unknown. Published microarray analysis suggested that succinate receptor 1 (Sucnr1) is specifically expressed in tuft cells. Thus, we hypothesized that the succinate–Sucnr1 axis may be utilized by tuft cells to detect certain infectious agents. Here we confirmed that Sucnr1 is specifically expressed in intestinal tuft cells but not in other types of intestinal epithelial cells, and demonstrated that dietary succinate induces tuft and goblet cell hyperplasia via Sucnr1 and the tuft cell-expressed chemosensory signaling elements gustducin and Trpm5. Conventional mice with a genetic Sucnr1 deficiency (Sucnr1
−/−) showed diminished immune responses to treatment with polyethylene glycol and streptomycin, which are known to enhance microbiota-derived succinate, but responded normally to inoculation with the parasitic worm Nippostrongylus brasiliensis that also produces succinate. Thus, Sucnr1 is required for microbiota-induced but not for a generalized worm-induced type 2 immunity.
Pyroptosis and inflammation are involved in the development of chronic obstructive pulmonary disease (COPD). However, the cigarette smoke-mediated mechanism of COPD remains unclear. In this study, we ...aimed to investigate the role of nucleotide-binding domain-like receptor protein-3 (NLRP3) inflammasome-mediated pyroptosis in the death of human bronchial epithelial (HBE) cells after cigarette smoke extract (CSE) exposure.
The protein level of NLRP3 in lung tissue was measured after cigarette smoke exposure in vivo. In vitro, HBE cells were treated with CSE. Subsequently, the activity of caspase-1, lactate dehydrogenase (LDH) release, release of interleukin (IL)-1β and NLRP3 expression levels were measured. The involvement of reactive oxygen species (ROS) was also explored.
After exposure to CSE, increased release of LDH, the transcriptional and translational upregulation of NLRP3, the caspase-1 activity levels, and enhanced IL-1β and IL-18 release were observed in 16HBE cells. In addition, NLRP3 was required to activate the caspase-1. Our results suggested that pre-stimulated of 16HBE with a caspase-1 inhibitor, or using NLRP3 siRNA to silence NLRP3 expression, also caused the decrease of IL-1β release and pyroptosis.
Significances.
CSE induced inflammation and contributed to pyroptosis through the ROS/NLRP3/caspase-1 pathway in 16HBE cells. The NLRP3 inflammasome participates in CSE-induced HBE cell damage and pyroptosis, which could provide new insights into COPD.
All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium ...adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium
. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.
Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies
. Here we studied 246,102 single epithelial cells from 16 ...early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8
alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8
cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.
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