Temporal single-cell atlas of non-neuronal retinal cells reveals dynamic, coordinated multicellular responses to central nervous system injury.

Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Neutrophils direct preexisting matrix to initiate repair in damaged tissues.

Internal organs heal injuries with new connective tissue, but the cellular and molecular events of this process remain obscure. By tagging extracellular matrix around the mesothelium lining in mouse peritoneum, liver and cecum, here we show that preexisting matrix was transferred across organs into wounds in various injury models. Using proteomics, genetic lineage-tracing and selective injury in juxtaposed organs, we found that the tissue of origin for the transferred matrix likely dictated the scarring or regeneration of the healing tissue. Single-cell RNA sequencing and genetic and chemical screens indicated that the preexisting matrix was transferred by neutrophils dependent on the HSF-integrin AM/B2-kindlin3 cascade. Pharmacologic inhibition of this axis prevented matrix transfer and the formation of peritoneal adhesions. Matrix transfer was thus an early event of wound repair and provides a therapeutic window to dampen scaring across a range of conditions.

CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment.

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture.

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

Stellate Cells in Tissue Repair, Inflammation, and Cancer.

Stellate cells are resident lipid-storing cells of the pancreas and liver that transdifferentiate to a myofibroblastic state in the context of tissue injury. Beyond having roles in tissue homeostasis, stellate cells are increasingly implicated in pathological fibrogenic and inflammatory programs that contribute to tissue fibrosis and that constitute a growth-permissive tumor microenvironment. Although the capacity of stellate cells for extracellular matrix production and remodeling has long been appreciated, recent research efforts have demonstrated diverse roles for stellate cells in regulation of epithelial cell fate, immune modulation, and tissue health. Our present understanding of stellate cell biology in health and disease is discussed here, as are emerging means to target these multifaceted cells for therapeutic benefit.

Dynamic changes to lipid mediators support transitions among macrophage subtypes during muscle regeneration.

Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we used mass spectrometry-based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed temporal regulation of glycerophospholipids and production of pro-inflammatory lipid mediators (for example, leukotrienes and prostaglandins) and specialized pro-resolving lipid mediators (for example, resolvins and lipoxins) that were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA sequencing of macrophages stimulated with resolvin D2 showed similarities to transcriptional changes found during the temporal transition from Ly6Chi macrophage to Ly6Clo macrophage. In vivo, resolvin D2 increased Ly6Clo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof of concept for their exploitable effector roles in muscle regeneration.

A Reservoir of Mature Cavity Macrophages that Can Rapidly Invade Visceral Organs to Affect Tissue Repair.

A key feature of inflammation is the timely recruitment of leukocytes, including monocytes, from blood into tissues, the latter maturing into macrophages over a period of 2-3 days. Using multi-channel spinning disk microscopy, we identified a rapid pathway of macrophage recruitment into an injured organ via a non-vascular route requiring no maturation from monocytes. In response to a sterile injury in liver, a reservoir of fully mature F4/80(hi)GATA6(+) peritoneal cavity macrophages rapidly invaded into afflicted tissue via direct recruitment across the mesothelium. The invasion was dependent on CD44 and DAMP molecule ATP and resulted in rapid replication and switching of macrophage toward an alternatively activated phenotype. These macrophages dismantled the nuclei of necrotic cells releasing DNA and forming a cover across the injury site. Rapid invasion of mature macrophages from body cavity with capacity for induction of reparative phenotype may impact altered tissues ranging from trauma to infections to cancer. VIDEO ABSTRACT.

Endogenous hydrogen sulfide production is essential for dietary restriction benefits.

Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK:

Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer.

Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.

Coordinated Spine Pruning and Maturation Mediated by Inter-Spine Competition for Cadherin/Catenin Complexes.

Dendritic spines are postsynaptic compartments of excitatory synapses that undergo dynamic changes during development, including rapid spinogenesis in early postnatal life and significant pruning during adolescence. Spine pruning defects have been implicated in developmental neurological disorders such as autism, yet much remains to be uncovered regarding its molecular mechanism. Here, we show that spine pruning and maturation in the mouse somatosensory cortex are coordinated via the cadherin/catenin cell adhesion complex and bidrectionally regulated by sensory experience. We further demonstrate that locally enhancing cadherin/catenin-dependent adhesion or photo-stimulating a contacting channelrhodopsin-expressing axon stabilized the manipulated spine and eliminated its neighbors, an effect requiring cadherin/catenin-dependent adhesion. Importantly, we show that differential cadherin/catenin-dependent adhesion between neighboring spines biased spine fate in vivo. These results suggest that activity-induced inter-spine competition for ß-catenin provides specificity for concurrent spine maturation and elimination and thus is critical for the molecular control of spine pruning during neural circuit refinement.

Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury.

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

The Cytokine TGF-ß Induces Interleukin-31 Expression from Dermal Dendritic Cells to Activate Sensory Neurons and Stimulate Wound Itching.

Cutaneous wound healing is associated with the unpleasant sensation of itching. Here we investigated the mechanisms underlying this type of itch, focusing on the contribution of soluble factors released during healing. We found high amounts of interleukin 31 (IL-31) in skin wound tissue during the peak of itch responses. Il31-/- mice lacked wound-induced itch responses. IL-31 was released by dermal conventional type 2 dendritic cells (cDC2s) recruited to wounds and increased itch sensory neuron sensitivity. Transfer of cDC2s isolated from late-stage wounds into healthy skin was sufficient to induce itching in a manner dependent on IL-31 expression. Addition of the cytokine TGF-ß1, which promotes wound healing, to dermal DCs in vitro was sufficient to induce Il31 expression, and Tgfbr1f/f CD11c-Cre mice exhibited reduced scratching and decreased Il31 expression in wounds in vivo. Thus, cDC2s promote itching during skin would healing via a TGF-ß-IL-31 axis with implications for treatment of wound itching.

Type 1 Innate Lymphoid Cells Protect Mice from Acute Liver Injury via Interferon-γ Secretion for Upregulating Bcl-xL Expression in Hepatocytes.

Although type 1 innate lymphoid cells (ILC1s) have been originally found as liver-resident ILCs, their pathophysiological role in the liver remains poorly investigated. Here, we demonstrated that carbon tetrachloride (CCl4) injection into mice activated ILC1s, but not natural killer (NK) cells, in the liver. Activated ILC1s produced interferon-γ (IFN-γ) and protected mice from CCl4-induced acute liver injury. IFN-γ released from activated ILC1s promoted the survival of hepatocytes through upregulation of Bcl-xL. An activating NK receptor, DNAM-1, was required for the optimal activation and IFN-γ production of liver ILC1s. Extracellular adenosine triphosphate accelerated interleukin-12-driven IFN-γ production by liver ILC1s. These findings suggest that ILC1s are critical for tissue protection during acute liver injury.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Injectable tissue prosthesis for instantaneous closed-loop rehabilitation.

To construct tissue-like prosthetic materials, soft electroactive hydrogels are the best candidate owing to their physiological mechanical modulus, low electrical resistance and bidirectional stimulating and recording capability of electrophysiological signals from biological tissues1,2. Nevertheless, until now, bioelectronic devices for such prostheses have been patch type, which cannot be applied onto rough, narrow or deep tissue surfaces3-5. Here we present an injectable tissue prosthesis with instantaneous bidirectional electrical conduction in the neuromuscular system. The soft and injectable prosthesis is composed of a biocompatible hydrogel with unique phenylborate-mediated multiple crosslinking, such as irreversible yet freely rearrangeable biphenyl bonds and reversible coordinate bonds with conductive gold nanoparticles formed in situ by cross-coupling. Closed-loop robot-assisted rehabilitation by injecting this prosthetic material is successfully demonstrated in the early stage of severe muscle injury in rats, and accelerated tissue repair is achieved in the later stage.

Injury prevents Ras mutant cell expansion in mosaic skin.

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3-12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.