White adipose tissue (WAT) is an essential regulator of energy storage and systemic metabolic homeostasis. Regulatory networks consisting of immune and structural cells are necessary to maintain WAT ...metabolism, which can become impaired during obesity in mammals. Using single-cell transcriptomics and flow cytometry, we unveil a large-scale comprehensive cellular census of the stromal vascular fraction of healthy lean and obese human WAT. We report new subsets and developmental trajectories of adipose-resident innate lymphoid cells, dendritic cells and monocyte-derived macrophage populations that accumulate in obese WAT. Analysis of cell-cell ligand-receptor interactions and obesity-enriched signaling pathways revealed a switch from immunoregulatory mechanisms in lean WAT to inflammatory networks in obese WAT. These results provide a detailed and unbiased cellular landscape of homeostatic and inflammatory circuits in healthy human WAT.
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.
Display omitted
•Activated liver ILC1s have a protective role in acute liver injury•DNAM-1 is required for activation and IFN-γ production of liver ILC1s•ATP accelerates IL-12-driven IFN-γ production by liver ILC1s•ILC1-derived IFN-γ helps the survival of hepatocytes through upregulation of Bcl-xL
Type 1 innate lymphoid cells (ILC1s) preferentially reside in liver, but the role of ILC1s in liver injury largely remains unknown. Nabekura and colleagues demonstrate that liver ILC1s have a protective role in acute liver injury, and ILC1-derived interferon-γ promotes the survival of damaged hepatocytes through upregulation of Bcl-xL.
Infection is restrained by the concerted activation of tissue-resident and circulating immune cells. Recent discoveries have demonstrated that tissue-resident lymphocyte subsets, comprised of innate ...lymphoid cells (ILCs) and unconventional T cells, have vital roles in the initiation of primary antiviral responses. Via direct and indirect mechanisms, ILCs and unconventional T cell subsets play a critical role in the ability of the immune system to mount an effective antiviral response through potent early cytokine production. In this review, we will summarize the current knowledge of tissue-resident lymphocytes during initial viral infection and evaluate their redundant or nonredundant contributions to host protection or virus-induced pathology.
CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate ...immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system.
Display omitted
•Optimized electroporation of cRNP complexes in primary mature innate immune cells•High knockout efficiency of signaling adaptors and transcription factors•cRNP-mediated knockout of Stat4 in primary NK cells during MCMV infection•cRNP knockout reveals MyD88 is required for cDC1-dependent control of MCMV infection
Riggan et al. optimize electroporation conditions for high-efficiency cRNP-mediated gene deletion in primary mature mouse innate immune cells and utilize this approach to elucidate gene function of NK cells and cDC1s using two in vivo adoptive transfer models during MCMV infection.
Tissue-resident immune cells are critical to the initiation and potentiation of inflammation. However, the tissue-protective cellular communication networks initiated by resident immunity during ...sterile inflammation are not well understood. Using single-cell transcriptomic analysis, we show the liver-resident cell connectome and signalome during acute liver injury. These analyses identify Il12b as a central regulator of liver injury-associated changes in gene expression. Interleukin (IL)-12 produced by conventional type 1 dendritic cells (cDC1s) is required for protection during acute injury through activation of interferon (IFN)-γ production by liver-resident type 1 innate lymphoid cells (ILC1s). Using a targeted in vivo CRISPR-Cas9 screen of innate immune sensing pathways, we find that cDC1-intrinsic cGAS-STING signaling acts upstream of IL-12 production to initiate early protective immune responses. Our study identifies the core communication hubs initiated by tissue-resident innate immune cells during sterile inflammation in vivo and implicates cDC1-derived IL-12 as an important regulator of this process.
Display omitted
•Il12b is a regulator of acute liver injury-induced changes in gene expression in silico•cDC1-derived IL-12 signaling protects against acute liver injury•cDC1-derived IL-12 induces protective liver-resident ILC1 IFN-γ responses•cDC1-intrinsic cGAS-STING signaling is required for IL-12 production
Hildreth et al. identify conventional type 1 dendritic cells (cDC1s) and interleukin (IL)-12 as critical regulators of sterile inflammation during acute liver injury. cDC1-intrinsic cGAS-STING signaling induces IL-12 to activate protective circuits including IFN-γ production by liver-resident type 1 innate lymphoid cells (ILC1s).
Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens ...directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.
CRISPR-Cas9 genome engineering can be used to functionally investigate the complex mechanisms of immune system regulation. Decades of work have aimed to genetically reprogram innate immunity, but ...current approaches are inefficient or nonspecific, limiting their use. Here, we detail an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells, resulting in high-efficiency editing of target gene expression from a single electroporation.
For complete details on the use and execution of this protocol, please refer to Riggan et al. (2020).
Display omitted
•Isolation of precursors and mature innate immune cells from peripheral tissues•Magnetic purification of group 1 innate lymphoid cells from spleen and liver•CRISPR RNP electroporation of primary innate immune cells•Analysis of gene editing efficiency via flow cytometry and Sanger sequencing
CRISPR-Cas9 genome engineering can be used to functionally investigate the complex mechanisms of immune system regulation. Decades of work have aimed to genetically reprogram innate immunity, but current approaches are inefficient or nonspecific, limiting their use. Here, we detail an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells, resulting in high-efficiency editing of target gene expression from a single electroporation.
Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of ...action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin‐derived compound design and testing that combines sequence‐free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20‐mer candidate peptides and scored them for charge, α‐helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical‐based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.
Obesity is associated with chronic low-grade white adipose tissue (WAT) inflammation that can contribute to the development of insulin resistance in mammals. Previous studies have identified ...interleukin (IL)-12 as a critical upstream regulator of WAT inflammation and metabolic dysfunction during obesity. However, the cell types and mechanisms that initiate WAT IL-12 production remain unclear. Here we show that conventional type 1 dendritic cells (cDC1s) are the cellular source of WAT IL-12 during obesity through analysis of mouse and human WAT single-cell transcriptomic datasets, IL-12 reporter mice and IL-12p70 protein levels by enzyme-linked immunosorbent assay. We demonstrate that cDC1s contribute to obesity-associated inflammation by increasing group 1 innate lymphocyte interferon-γ production and inflammatory macrophage accumulation. Inducible depletion of cDC1s increased WAT insulin sensitivity and systemic glucose tolerance during diet-induced obesity. Mechanistically, endocytosis of apoptotic bodies containing self-DNA by WAT cDC1s drives stimulator of interferon genes (STING)-dependent IL-12 production. Together, these results suggest that WAT cDC1s act as critical regulators of adipose tissue inflammation and metabolic dysfunction during obesity.
Abstract
CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. ...While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.
PDF
