IL-4 and IL-13: Regulators and Effectors of Wound Repair.

Type 2 immunity mediates protective responses to helminths and pathological responses to allergens, but it also has broad roles in the maintenance of tissue integrity, including wound repair. Type 2 cytokines are known to promote fibrosis, an overzealous repair response, but their contribution to healthy wound repair is less well understood. This review discusses the evidence that the canonical type 2 cytokines, IL-4 and IL-13, are integral to the tissue repair process through two main pathways. First, essential for the progression of effective tissue repair, IL-4 and IL-13 suppress the initial inflammatory response to injury. Second, these cytokines regulate how the extracellular matrix is modified, broken down, and rebuilt for effective repair. IL-4 and/or IL-13 amplifies multiple aspects of the tissue repair response, but many of these pathways are highly redundant and can be induced by other signals. Therefore, the exact contribution of IL-4Rα signaling remains difficult to unravel.

The extracellular matrix integrates mitochondrial homeostasis.

Cellular homeostasis is intricately influenced by stimuli from the microenvironment, including signaling molecules, metabolites, and pathogens. Functioning as a signaling hub within the cell, mitochondria integrate information from various intracellular compartments to regulate cellular signaling and metabolism. Multiple studies have shown that mitochondria may respond to various extracellular signaling events. However, it is less clear how changes in the extracellular matrix (ECM) can impact mitochondrial homeostasis to regulate animal physiology. We find that ECM remodeling alters mitochondrial homeostasis in an evolutionarily conserved manner. Mechanistically, ECM remodeling triggers a TGF-ß response to induce mitochondrial fission and the unfolded protein response of the mitochondria (UPRMT). At the organismal level, ECM remodeling promotes defense of animals against pathogens through enhanced mitochondrial stress responses. We postulate that this ECM-mitochondria crosstalk represents an ancient immune pathway, which detects infection- or mechanical-stress-induced ECM damage, thereby initiating adaptive mitochondria-based immune and metabolic responses.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.

Disruption of cellular plasticity by repeat RNAs in human pancreatic cancer.

Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single-cell resolution in 46 primary tumors, revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF responses are driven by distinct innate immune signaling through interferon regulatory factor 3 (IRF3). The cell-context-specific viral-like responses to repeat RNAs provide a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment.

Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

Extracellular cytochrome nanowires appear to be ubiquitous in prokaryotes.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

Engineering RNA export for measurement and manipulation of living cells.

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.

Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Reciprocal cell-ECM dynamics generate supracellular fluidity underlying spontaneous follicle patterning.

During vertebrate embryogenesis, cell collectives engage in coordinated behavior to form tissue structures of increasing complexity. In the avian skin, assembly into follicles depends on intrinsic mechanical forces of the dermis, but how cell mechanics initiate pattern formation is not known. Here, we reconstitute the initiation of follicle patterning ex vivo using only freshly dissociated avian dermal cells and collagen. We find that contractile cells physically rearrange the extracellular matrix (ECM) and that ECM rearrangement further aligns cells. This exchange transforms a mechanically unlinked collective of dermal cells into a continuum, with coherent, long-range order. Combining theory with experiment, we show that this ordered cell-ECM layer behaves as an active contractile fluid that spontaneously forms regular patterns. Our study illustrates a role for mesenchymal dynamics in generating cell-level ordering and tissue-level patterning through a fluid instability-processes that may be at play across morphological symmetry-breaking contexts.

Extracellular pH sensing by plant cell-surface peptide-receptor complexes.

The extracellular pH is a vital regulator of various biological processes in plants. However, how plants perceive extracellular pH remains obscure. Here, we report that plant cell-surface peptide-receptor complexes can function as extracellular pH sensors. We found that pattern-triggered immunity (PTI) dramatically alkalinizes the acidic extracellular pH in root apical meristem (RAM) region, which is essential for root meristem growth factor 1 (RGF1)-mediated RAM growth. The extracellular alkalinization progressively inhibits the acidic-dependent interaction between RGF1 and its receptors (RGFRs) through the pH sensor sulfotyrosine. Conversely, extracellular alkalinization promotes the alkaline-dependent binding of plant elicitor peptides (Peps) to its receptors (PEPRs) through the pH sensor Glu/Asp, thereby promoting immunity. A domain swap between RGFR and PEPR switches the pH dependency of RAM growth. Thus, our results reveal a mechanism of extracellular pH sensing by plant peptide-receptor complexes and provide insights into the extracellular pH-mediated regulation of growth and immunity in the RAM.

ER stress transforms random olfactory receptor choice into axon targeting precision.

Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.

Tissue Morphogenesis Through Dynamic Cell and Matrix Interactions.

Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but also serve as key regulators of tissue morphogenesis. Cells constantly probe their environment to make decisions: They integrate chemical and mechanical information from the environment via diffusible ligand- or adhesion-based signaling to decide whether to release specific signaling molecules or enzymes, to divide or differentiate, to move away or stay, or even whether to live or die. These decisions in turn modify their environment, including the chemical nature and mechanical properties of the extracellular matrix. Tissue morphology is the physical manifestation of the remodeling of cells and matrices by their historical biochemical and biophysical landscapes. We review our understanding of matrix and adhesion molecules in tissue morphogenesis, with an emphasis on key physical interactions that drive morphogenesis.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function.

Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

The Immunoglobulin Superfamily Receptome Defines Cancer-Relevant Networks Associated with Clinical Outcome.

Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome. Using a high-throughput technology to interrogate most single transmembrane receptors for binding to 445 IgSF proteins, we identify over 500 interactions, 82% previously undocumented, and confirm more than 60 receptor-ligand pairs using orthogonal assays. Our study reveals a map of cell-type-specific interactions and the landscape of dysregulated receptor-ligand crosstalk in cancer, including selective loss of function for tumor-associated mutations. Furthermore, investigation of the IgSF interactome in a large cohort of cancer patients identifies interacting protein signatures associated with clinical outcome. The IgSF interactome represents an important resource to fuel biological discoveries and a framework for understanding the functional organization of the surfaceome during homeostasis and disease, ultimately informing therapeutic development.

Microglial Remodeling of the Extracellular Matrix Promotes Synapse Plasticity.

Synapse remodeling is essential to encode experiences into neuronal circuits. Here, we define a molecular interaction between neurons and microglia that drives experience-dependent synapse remodeling in the hippocampus. We find that the cytokine interleukin-33 (IL-33) is expressed by adult hippocampal neurons in an experience-dependent manner and defines a neuronal subset primed for synaptic plasticity. Loss of neuronal IL-33 or the microglial IL-33 receptor leads to impaired spine plasticity, reduced newborn neuron integration, and diminished precision of remote fear memories. Memory precision and neuronal IL-33 are decreased in aged mice, and IL-33 gain of function mitigates age-related decreases in spine plasticity. We find that neuronal IL-33 instructs microglial engulfment of the extracellular matrix (ECM) and that its loss leads to impaired ECM engulfment and a concomitant accumulation of ECM proteins in contact with synapses. These data define a cellular mechanism through which microglia regulate experience-dependent synapse remodeling and promote memory consolidation.