Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults [preprint]

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection

The Regulation of Immunological Processes by Peripheral Neurons

The nervous system and the immune system are the primary sensory interfaces between the internal and external environment. They are responsible for recognizing, integrating, and responding to stimuli with the appropriate valence and magnitude to optimize host fitness. Furthermore, an alluring parallel concept is that both systems have the capacity to form memories of these encounters leading to optimized and adaptive future responses. Recent work in the fields of neuroscience and immunology has led to a high-resolution map of cell subsets within both systems. Here, we start to leverage these advances to explore the relationship between these two sensory systems at the level of discrete cell subsets in immunological disease and homeostasis with a focus on interactions at barrier tissues. This work stems from the initial hypothesis that sensory neurons for noxious stimuli, nociceptors, regulate inflammation by controlling production of key instructive and effector cytokines derived from tissue-resident immune cells. In our first investigation focused on the skin, we identified that NaV1.8+ TRPV1+ nociceptors, via interactions with dDCs, are essential in vivo regulators of interleukin-(IL)-23/IL-17 pathway cutaneous immune responses. This set of studies raised several intriguing questions including determining what the inputs and outputs of nociceptors are that regulate IL-23 production from dDCs and whether nociceptors play a role in distinct inflammatory contexts. In further experiments we determined that while TRPV1 itself is dispensable, nociceptor activity is essential to promote disease and local cytokine production. As we found that nociceptors are critical for cutaneous IL-23 production, we tested the effect of nociceptor ablation during a bacterial infection that depends on IL-23 for clearance. Surprisingly, the absence of NaV1.8+ nociceptors led to more severe skin pathology and bacterial dissemination suggesting that NaV1.8+ nociceptors are important for tissue protection and bacterial containment. In complementary studies assessing the role of nociceptors in atopic dermatitis (Type 2 inflammation), we found that TRPV1+ neurons did not regulate gross disease or the instructive cytokine thymic stromal lymphopoietin (TSLP), yet were required for full effector (IL-4) cytokine production. This suggests that nociceptors exert contextual specificity and are not exclusively pro-inflammatory in all cutaneous pathologies. Based on the role for nociceptors in regulating cutaneous IL-23 and subsequent inflammation, we hypothesized that TRPV1+ nociceptors would be important in regulating IL-23-driven pathologies in the gut. However, we found no appreciable role for nociceptors in either an acute innate colitis or a spontaneous microbiota-driven colitis. While colitis is characterized by its gastrointestinal pathology, it affects the body systemically and we noted that in both models, mice lacking TRPV1+ nociceptors maintained healthier weights than their nerve-replete counterparts. This opens future avenues for exploration on how sensing of inflammation by nociceptors influences microbial communities, systemic metabolism, and sickness behavior. In summary, we propose that sensory perception of inflammation by peripheral neurons exerts context-dependent effects on immunological processes. We speculate that there is the potential to uncover a modular design framework by which distinct lines of sensory neurons are triggered in specific settings and may contribute to regulating different aspects of inflammation through interactions with immune cell subsets.Medical Science

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4⁺ enterocytes, microfold-like cells, and IL13RA2⁺IL11⁺ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.National Institutes of Health (U.S.) (Grant 5U24AI118672

Allergic inflammatory memory in human respiratory epithelial progenitor cells

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases [superscript 1]. Specialized subsets of epithelial cells—including secretory and ciliated cells—differentiate from basal stem cells to collectively protect the upper airway [superscript 2–4]. Allergic inflammation can develop from persistent activation [superscript 5] of type 2 immunity [superscript 6] in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps [superscript 7]. Basal cell hyperplasia is a hallmark of severe disease [superscript 7–9], but it is not known how these progenitor cells [superscript 2,10,11] contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing [superscript 12], report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic [superscript 13], epigenetic [superscript 14,15] and extrinsic factors [superscript 11,16,17] that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories. KNational Institutes of Health (U.S.) (Grant 1DP2OD020839)National Institutes of Health (U.S.) (Grant 2U19AI089992)National Institutes of Health (U.S.) (Grant 1U54CA217377)National Institutes of Health (U.S.) (Grant P01AI039671)National Institutes of Health (U.S.) (Grant 5U24AI118672)National Institutes of Health (U.S.) (Grant 2RM1HG006193)National Institutes of Health (U.S.) (Grant 1R33CA202820)National Institutes of Health (U.S.) (Grant 2R01HL095791)National Institutes of Health (U.S.) (Grant 1R01AI138546)National Institutes of Health (U.S.) (Grant 1R01HL126554)National Institutes of Health (U.S.) (Grant 1R01DA046277)National Institutes of Health (U.S.) (Grant 2R01HL095791)Bill & Melinda Gates Foundation (Grant OPP1139972)Bill & Melinda Gates Foundation (Grant OPP1116944)National Institutes of Health (U.S.) (Grant 2R01GM081871–09 )National Cancer Institute (U.S.) (Grant P30-CA14051)National Institutes of Health (U.S.). Center for AIDS Research (Award P30 AI060354

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5⁺ ISCs. We show that MHCII⁺ Lgr5⁺ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4⁺ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5⁺ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5⁺ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5⁺ ISCs, thus, orchestrate tissue-wide responses to external signals. Intestinal stem cells act as non-conventional antigen presenting cells, and these interactions with T helper cells modulate ISC renewal and differentiation to shape the intestine. Keywords: gut biology; intestinal stem cells; ISCs; T helper; Th; mucosal immunity; MHC class II; MHCII; tuft cells; T regulatory; Treg; single cell RNA-seq; scRNA-seq; epithelial differentiation; stem cell renewalNational Institute of Health (U.S.) (Award 1DP2OD020839)National Institute of Health (U.S.) (Grant CA211184)National Institute of Health (U.S.) (Grant AG045144

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection