Epigenetics and tissue immunity—Translating environmental cues into functional adaptations *

There is an increasing appreciation that many innate and adaptive immune cell subsets permanently reside within non‐lymphoid organs, playing a critical role in tissue homeostasis and defense. The best characterized are macrophages and tissue‐resident T lymphocytes that work in concert with organ structural cells to generate appropriate immune responses and are functionally shaped by organ‐specific environmental cues. The interaction of tissue epithelial, endothelial and stromal cells is also required to attract, differentiate, polarize and maintain organ immune cells in their tissue niche. All of these processes require dynamic regulation of cellular transcriptional programmes, with epigenetic mechanisms playing a critical role, including DNA methylation and post‐translational histone modifications. A failure to appropriately regulate immune cell transcription inevitably results in inadequate or inappropriate immune responses and organ pathology. Here, with a focus on the mammalian kidney, an organ which generates differing regional environmental cues (including hypersalinity and hypoxia) due to its physiological functions, we will review the basic concepts of tissue immunity, discuss the technologies available to profile epigenetic modifications in tissue immune cells, including those that enable single‐cell profiling, and consider how these mechanisms influence the development, phenotype, activation and function of different tissue immune cell subsets, as well as the immunological function of structural cells.

to ensure appropriate immune responses are generated and terminated in a timely manner, and a failure to do so may have negative consequences for organ homeostasis. Therefore, understanding the precise epigenetic mechanisms that control tissue immune responses will inform treatment strategies for a variety of diseases. Here, with a focus on the mammalian kidney, we will review the basic concepts of tissue immunity, discuss the technologies available to profile epigenetic modifications in tissue immune cells, and consider how these mechanisms influence the development, phenotype and function of different tissue immune cell subsets, as well as the immunological function of structural cells in health and disease.

| Tissue immunity-a coordinated effort by structural cells and tissue-resident immune cells
The study of mammalian immunity has historically focused on interrogating the responses of immune cells in blood or secondary lymphoid organs (lymph node and spleen). However, it is increasingly appreciated that several subsets of innate and adaptive immune cells reside in non-lymphoid organs. [1][2][3] These tissue-resident populations may constitute a large proportion of the total immune cell pool, and do not enter the circulation, permanently occupying a specific niche within tissues. 4 The archetypal tissue-resident cell type is the macrophage, exemplifying the canonical features of tissue-resident cells, being long-lived, self-renewing, and showing tissue-specific transcriptional and functional specialization. 5 Macrophages take up residency in tissue niches early in embryogenesis, seeding from the yolk sac (YS), and then fetal liver precursors. 6 Post-natally, macrophage tissue pools are variably replenished by monocyte-derived cells, 7,8 with tissue-specific cues, for example from the microbiome 8 (in the case of the intestine) or high interstitial sodium 9 (in the case of the kidney), influencing this process. Other prevalent tissue-resident immune cell subsets include T cells; antigen-specific CD8 + T and CD4 + T cells enter tissues during viral challenge, and persist long after the resolution of infection. [10][11][12] A common tissue-residency transcriptional signature has been described in lymphocytes, 13,14 and other tissue-resident subsets including innate lymphocytes and natural killer (NK) cells have also been characterized. 15,16 Tissue-resident immune cells play a variety of important functional roles in addition to immune defense, frequently contributing to organ homeostasis. [17][18][19] For example, human yolk sac-derived macrophages in the heart were physically connected to cardiomyocytes via gap-junctions containing connexin 43, which allows macrophages to participate in and regulate electrical conduction 20 ; in the colon, muscularis macrophages regulate peristalsis. 21 Effective tissue immune responses require the coordinated interaction of these resident populations with each other and with their circulating counterparts, via cytokine and chemokine production, as well as cross-talk with the epithelial compartment. [22][23][24][25] Indeed, immune functionality within organs is not limited to immune cells, but non-immune tissue cells can also play a part. For example, in human and mouse kidney, we previously showed that pelvic epithelial cells express antimicrobial peptides (AMP), directly contributing to anti-bacterial immunity, as well as producing neutrophil-recruiting chemokines, orchestrating the specific anatomical localization of the key circulating phagocytes to protect the kidney from bacteria ascending from the bladder. 26 Krausgruber et al 27 described the expression of immune mediators, as well as cytokines and chemokines in epithelium, fibroblast and endothelium, in a variety of mouse organs, generating so-called 'structural immunity'. Thus, tissue immune responses involve the combined efforts and interactions of epithelial, endothelial, stromal, and resident immune cells, and are tailored to the tissue-specific challenges encountered, requiring tissue-specific cues to control cellular transcriptional programmes.

| Experimental identification of tissue immune cells
CD49a. [31][32][33][34] In humans, assessing tissue residency is more challenging, but T cells isolated from non-lymphoid organs express some of these markers 35,36 ; For example, CD69 is detectable on skin-resident T cells in humans. 37,38 However, there are differences in the phenotypes of Trm in murine versus human organs 38 and different organs imprint distinct tissue-specific transcriptional programmes, phenotypes, and functions on resident T cells. 35,39 To summarize, identifying and studying bona fide tissue-resident subsets requires careful application of the experimental systems discussed above, and is particularly challenging in humans, but is necessary to definitively delineate the organ-specific transcriptomic and epigenetic profiles of resident immune cells.

| Tissue cues, structural and immune cells in the kidney
Every organ presents a unique environment for the immune cells residing there, with specific tissue cues, shaped by the homeostatic function of the organ. In many cases, there are discrete microenvironments within an organ, due to spatial separation of different organ functions. A good example of this is found in the mammalian kidney, an organ specialized for the removal of metabolic waste and excess fluid. Anatomically, each kidney consists of an outer cortex containing glomeruli where filtrate is generated, and an inner medulla where urine is concentrated ( Figure 1A). The functional subunit of the kidney is the nephron, made up of a glomerulus, proximal tubule (PT) (where filtered electrolytes are reabsorbed), loop of Henle (LOH) (that generates the intrarenal sodium gradient required for urine concentration), and collecting ducts (CD) that coalesce in the kidney pelvis. 40 Different mononuclear phagocyte (MNP) populations are differentially located in cortex and medulla. 39 Furthermore, immune cells in the cortex are exposed to a very different environment compared to medullary immune cells that experience hypersalinity and hypoxia. 41 Notably, these environmental cues can affect immune cell recruitment and function; we found that high extracellular sodium augmented the anti-bacterial function of macrophages, and increased the production of monocyte-recruiting chemokines by epithelial cells, 40 effects mediated at a molecular level by the transcription factors Nuclear Factor Of Activated T Cells 5 (NFAT5) and Hypoxia Inducible Factor 1 Subunit Alpha (HIF1ɑ). 42 We recently applied single-cell RNA sequencing (scRNAseq) to more comprehensively profile kidney immune cells, 26 utilizing technological advances that have enabled highthroughput scRNAseq and the generation of organ atlases. 43 In normal human kidney, we identified more than 15

| EPIG ENE TIC MECHANIS MS
Epigenetic mechanisms play a crucial role in cell fate specification by regulating gene expression and silencing in a context-dependent manner. Epigenetic control of gene transcription and translation does not involve changes to the DNA sequence, but rather, reversible chemical modifications of DNA or histones, or the activities of non-coding RNAs, that together enable cell-and tissue-specific, gene expression patterns that are essential for controlling normal developmental processes and maintaining tissue homeostasis.
Disruption of epigenetic mechanisms can lead to organ dysfunction and disease states, including autoimmunity and cancer.

| DNA methylation
DNA methylation is one of the best-studied epigenetic modifications to date. In general, DNA methylation leads to the addition of a methyl group to the fifth carbon atom of a cytosine (5mC) followed by a guanine base ( Figure 2A). As a result, the methylated CpG dinucleotides, which are frequently found in gene regulatory regions, block transcription factor binding to gene promoters, and repress target gene expression. Currently, three active DNA methyltransferases (DNMTs) are known to catalyze DNA methylation in mammals, namely DNMT1, DNMT3A, and DNMT3B. 44 Demethylation in the mammalian genome is mediated by the TET (Ten-Eleven Translocation) family of dioxygenases that oxidize 5mC to 5-hydroxymethylcytosine (5hmC), and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 45 The "intermediate" 5hmC marks active demethylation, plays distinct epigenetic roles, and is a useful indicator of gene expression. 46,47 DNMT1 is essential for maintaining DNA methylation at the synthesis phase of cell cycle, 48 while DNMT3A and DNMT3B are required for de novo DNA methylation. During embryogenesis, DNA is first demethylated by TET1, TET2, and TET3, resulting in a "clean slate" for de novo DNA methylation by DNMT3A and DNMT3B. 49 The activities of these two enzymes are regulated by their expression pattern and structure of distinct isoforms; DNMT3A has two isoforms DNMT3A1 and DNMT3A2. 50 Full-length DNMT3A1 expression is maintained in differentiated cells and its intact Nterminal region can interact with DNA to repress gene expression. In contrast, DNMT3A2, which lacks 223 amino acids in its N-terminal region, is predominantly expressed in embryonic stem cells (ESC).
DNMT3B has more than 30 isoforms with distinct catalytic and regulatory activities. Although loss-of-function studies of DNMT3A and DNMT3B confirmed their importance in de novo methylation rather than imprinted methylation patterns, 51  F I G U R E 1 Tissue immunity in the human kidney. A, The human kidney in section; the kidney is macroscopically divided into cortex and medulla. Hundreds of thousands of nephron units are arranged over cortico-medullary depth (highlighted). Filtrate is generated in the glomerulus (lower panel) comprising podocytes, mesangial cells, and glomerular endothelial cells (GEC), before being modified by solute and metabolite resorption, excretion, and concentration along tubular nephron segments. Gradients of oxygen tension and salinity exist between the cortex and medulla as indicated. The generation of functionally specialized, differentiated immune cell subsets is regulated by DNA methylation, for example, regulatory T cell (Treg) differentiation is coordinated by DNMT1, 56 T helper (Th)1/2/17 polarization by TET2 and DNMT3A, 57,58 and memory T cell activation by demethylation of interferon gamma (IFNG). 59 These loss-of-function studies have not only revealed the role of these enzymes in methylation or demethylation of specific CpG sites, but also show their influence in controlling the activity of transcription factors that may bind to enhancers in these cell types.  Argonaute proteins to achieve target gene silencing, 79 particularly of transposons. 80 The biogenesis of piRNAs is not yet clear; however, it is thought that they could be derived from long, single-stranded precursor molecules, 81 catalyzed by two piwi proteins Aubergine (Aub) and Argonaute-3 (Ago3). This process, also known as the piRNA Ping-Pong pathway, appears to trigger the degradation of transposons. 82 lncRNAs transcripts exceed 200 nucleotides in length, for example, X-inactive specific transcript (XIST), a 17kb lncRNA best known for its role in X chromosome inactivation. XIST physically binds to its target X chromosome in cis, recruiting the Polycomb complex 2 (PRC2). 82 As a result, H3K27 trimethylation is induced to repress gene expression.

| DNA methylation profiling
Genome-wide assays of DNA methylation (5mC) can be performed using methods which capture the entire genome at single-base resolution, or methods that target specific modifications and regions of the genome to build lower resolution maps of methylation ( Figure 2B).
The current gold standard is whole-genome bisulfite sequencing (WGBS). A high concentration of sodium bisulfite at pH 5.0 results in deamination of cytosine to uracil, while 5mC is protected from the deamination reaction. 83 Consequently in sequencing data, 5mC are read as cytosine bases; however, the deaminated bases are sequenced as thymines. In the original methodology, Sanger sequencing was used to assess CpG methylation. 83,84 The current standard approach is to prepare libraries for next-generation sequencing (NGS) to generate genome-scale maps of DNA methylation. 85 WGBS cannot differentiate 5mC and 5hmC-both are read as cytosines in sequencing. A modification to bisulfite sequencing, oxidative bisulfite sequencing (OxBS-seq), converts 5hmC to 5fC, and subsequent bisulfite treatment converts 5fC to uracil, leaving 5mC unconverted. Comparisons between oxBS-seq and conventional bisulfite sequencing allow for identification of 5hmC modified regions. 86,87 An alternative method for 5hmC profiling utilizes differential TET enzyme-mediated oxidation; in this assay, 5hmC is converted to β-glucosyl-5-hydroxymethylcytosine (g5hmC) by β-glucosyltransferase, and is protected from oxidation by TET.
Consequently, 5hmC is sequenced as cytosine after bisulfite conversion, whereas 5mC which becomes oxidized to 5caC is sequenced as thymine. 88,89 Protocols for single-cell bisulfite sequencing (scBS-seq) have been developed, consisting of single-cell isolation into plate wells, prior to bisulfite conversion and library construction. 90 As a proof of principle, these methods have been used to interrogate DNA methylation patterns in ESC. 91 Using an affinity purification approach, 5mC specific antibodies 105 or methyl-CpG binding protein 106 can be used to enrich regions of the genome which are highly methylated. After immunoprecipitation, fragments enriched for 5mC can be assayed by array hybridization or next-generation sequencing. This method is highly economical but biases toward hypermethylated CpG rich regions of the genome. 107 A further method providing a low-cost and high-throughput view of the methylome uses bisulfite conversion of genomic DNA followed by PCR amplification and hybridization to a microarray. This method-the Illumina HMEPIC BeadChips generate data on 850 000 methylation sites across the genome, and build on the 450 000 site HMK450 chip predecessor. 108 Although this does not provide singlebase resolution, it does offer coverage of the 95% of CpG islands and is well suited to high-throughput approaches assaying methylation variation in population studies.
Long-read methods including nanopore sequencing, and SMRT sequencing have also been used to assay methylation status genome-wide without requiring bisulfite conversion via picoampere signal intensities corresponding to modified bases (Nanopore), 109 or variation in polymerase kinetic activity (SMRT sequencing). 110 Although these methods generate data with a higher error rate and modest throughput, they are able to provide much longer reads allowing more efficient interrogation of methylome haplotypes 111 and delineation of methylation status at repetitive elements and structural variants. 112

| Histone modifications
Genome-wide profiles of chromatin modifications can be routinely assayed using chromatin immunoprecipitation followed by sequencing (ChIP-seq) ( Figure 2B). This method utilizes protein affinity purification using an antibody specific for a chromatin post-translational modification or another DNA-binding protein such as a transcription factor. Following crosslinking of DNA-protein complexes, fragmentation, and exonuclease treatment, DNA-protein complexes are immunoprecipitated, and enriched DNA fragments are sequenced using NGS. [113][114][115][116] This method has been extremely widely used to profile genomic regions associated with transcription factors, and a broad range of histone acetylation and methylation states. types. 133,134 Omni-ATAC also worked on frozen sample blocks, which were historically difficult to assay. 133 Fast-ATAC is an optimized ATACseq protocol for blood cells, which produced highquality data with reduced noise. 134 ATACseq has been adapted to a high-throughput single-cell method, initially as a nano-well based method, 135 before its evolution to a massively parallel dropletmicrofluidics implementation capable of profiling hundreds of thousands of cells simultaneously. 136 Initial work using this method has illustrated its utility in studying dynamic developmental processes, chiefly epigenomic differentiation trajectories during haematopoeisis. 135,136 Integrating genomic variants associated with blood cell traits, with regions of accessible chromatin during hematopoiesis, has allowed investigators to probe genome regulation across blood cell lineages. 137 scATACseq has also found utility in distinguishing clonal relationships between cells via mitochondrial mutation tracing. As the mitochondrial genome is non-chromatinized, scATACseq (but not snATACseq) generates abundant reads at high coverage across the

| Chromatin conformation
The three-dimensional organization of the genome within the nucleus can bring distant domains close together and can act as an important set of regulatory mechanisms. 140 These structures include the 30 nm fiber, chromatin loops, and interchromosomal interactions. The systematic study of genome topology has been enabled by Chromosome Conformation Capture (3C) ( Figure 2B). In this method, interactions between distant loci are captured by formaldehyde crosslinking of chromatin. Thereafter DNA is digested by restriction enzymes generating crosslinked fragments, which are then re-ligated forming chimeric DNA molecules for subsequent PCR of target loci. 141 The development of this technology has spawned two decades of adapted techniques using sequencing to capture chromatin organization, including 4C, 5C and, capture-C, 142 and HiC. 143,144 Similarly to the original 3C method, HiC generates chimeric DNA fragments following formaldehyde crosslinking. These are subjected to paired end sequencing, before mapping to the genome, followed by computation identification of higher-order interactions such as chromatin loops and Topologically Associating Domains (TADs). HiC is therefore able to uncover the contact probabilities of DNA across the entire genome. This method has been applied to the study of immunity, investigating dynamic remodeling of chromatin conformation during T cell development and activation. [145][146][147] HiC has been extended to a single-cell application, allowing the

| EPI G ENE TI C CONTROL OF TISSUE IMMUNE CELL S
Given the importance of epigenetics in controlling cell fate specification and subsequent function, delineating tissue-specific epigenetic controllers in resident immune cells has gained increasing interest.
The development of robust assays (discussed in Section 4) has enabled key mechanisms controlling context-specific gene expression to be profiled for the major tissue-resident immune cell populations, which will be discussed in turn.

| Macrophages
Macrophages are critical tissue immune sentinels and regulate many well as in humanized mice. 166 Indeed, in human adult kidneys, we defined transcriptomically distinct clusters of mononuclear phagocytes (MNPs) as tissue-resident macrophages or monocyte-derived macrophages based on transcriptional similarity to human fetal kidney macrophages, as well as fate-mapped YS macrophages in mouse. 26 In mice, transitioning of short-lived Ly6C high MHC-II low (major histocompatibility complex 2) monocytes to mature Ly6C low MHC-II high monocytes was found to be dependent on CCAAT-enhancer-binding protein beta (C/EBPβ), which binds to the promoter of Nr4a1 (also known as Nurr77) and induced its expression, 169   In addition, differentiation from monocytes to macrophages requires signaling from the cytokine colony-stimulating factor 1 (CSF1) receptor (CSF1R). 170 Genetic deletion or antibody-mediated depletion of CSF1 leads to macrophage deficiency in adult tissues. 171,172 Cells that produce CSF1 provide a niche for macrophage homeo- for example, ATACseq peaks for FIRE in lung and intestinal macrophages were much lower than that observed in brain microglia, suggesting that chromatin accessibility for FIRE is relatively greater in microglia. 182,183 The transition of human monocytes to macrophages is also dependent on the regulation of the epigenome. For instance, the BLUEPRINT consortium performed genome-wide epigenome profiling (via DNase I-seq, ChIP-seq, and RNA-seq) on monocytes extracted from human peripheral blood, as well as macrophages, generated ex vivo from monocytes, cultured in the presence or absence of various stimuli. 184 They found that ~8000 regions were hypersensitive to DNase I cleavage and marked by differential his- For instance, the acquisition of a macrophage program was shown to occur in tandem with organogenesis where dynamic spatial and temporal expression of key tissue-specific transcriptional regulators defined the diversity and heterogeneity of macrophages in developing mice. 5 As such, the transcriptional regulatory landscape of tissue-resident macrophages is a balance between nurture (environment) and nature (ontological lineage). Combining RNA sequencing, genome-wide ChIP-seq and ATACseq on macrophages, monocytes, and granulocytes showed broad cell type-specific histone modifications on specific enhancer elements unique to each group, for example, H3K4me3 marks were present on the promoter of Mertk in macrophages but not monocytes or neutrophils. 183 Furthermore, the enhancer landscape in tissue-resident macrophages was also distinct depending on the tissue/environment, as well as ontogeny 183 ( Figure 4B). In a separate example, the epigenetic landscape governing KC identity and diversity in the liver is determined by spatially distinct microanatomical niches in the liver sinusoids. 186 This was associated with similar patterns of chromatin accessibility between KC and non-alcoholic steatohepatitis (NASH)-associated Tim4 -KClike recruited macrophages, which occupy similar spatial locations, compared to Ly6c2 expressing monocyte-related macrophages. 186 Tissue macrophages are also regulated by distinct migration cues instructed by the environment. For instance, in the kidneys, we previously showed that high interstitial sodium in the medullary/pelvic zones induced CCL2 and C-X3-C Motif Chemokine Ligand 1 (CX3CL1) chemokine production by medullary tubular epithelial cells, which in turn recruits CCR2/CX3CR1-expressing CD14 + monocytes 9 ( Figure 5). Perturbation to the renal sodium gradient, for example, in patients with diabetes insipidus, a condition where urine concentration is impaired 187 due to reduced secretion/action of the antidiuretic hormone, vasopressin, there was reduced recruitment of monocytes to the medulla. 9 In a reciprocation fashion, in the Dahl salt-sensitive rat model, a pre-clinical model for salt-dependent hypertension and chronic kidney disease, 188 increased infiltration of macrophages,

T cells, and B cells was observed. Abnormalities of tissue immune
cells have also been reported with a high-salt diet, 189 including an increased ratio of CD86 + to CD163 + macrophages in the kidney, interpreted as an increased M1:M2 ratio. 190 Overall, salt-related effects were shown in multiple studies to be mediated by NFAT5, a key transcription factor that senses hypertonicity and regulates gene expression to enable cellular adaptation to hyperosmotic stress 191,192 ; infection. 193   This, in turn, activated Nfat5, as a downstream signaling target from the p38/MAPK pathway, 194,195 and expression of Nos2, boosting the nitric oxide-mediated Leishmanicidal activity. 193  and Tcf4. 249,252,253 Similarly, in a separate ChIP-seq study comparing moDCs versus pDCs, while there was relative small number of differentially enriched activating promoter H3K4Me3 marks, there was a substantial differential usage of H3K4me1 and H3K27ac enhancer marks between the two cell types. 254 In particular, they found that Irf8 and Cebpb bound more regions and pDC-or moDC-specific H3K4Me1 enhancer regions in pDC and moDC respectively. 254 shRNA knockdown and overexpression systems of Irf8 or Cebpb (whole-genome shotgun bisulfite sequencing). 255 Although only a small percentage of hypomethylation events were situated near a promoter region, with the majority in intergenic or intronic regions, a large proportion of regions were situated within H3K4me1 primed enhancer regions already present in non-infected DCs. 255 Live MTB infection increased association with active H3K27ac marks and decreased association with H3K4me1 (primed) marks. 255 Infection was associated with increased chromatin accessibility of active enhancer marks, as well as increased genome-wide binding of NF-κB and IRF transcription factors in infected DCs. 245 However, a subsequent study in the same DC infection model found that changes in gene expression were not entirely dependent on methylation status, 246 suggesting that they play a role in fine-tuning immune responses or even innate immune memory.
During autoimmune disease, for example in lupus nephritis, circulating DCs (CD1c + moDCs and pDCs) showed increased mRNA expression of IRF1 and IRF8 in disease compared to control, in moDCs and pDCs respectively. 257 There was a trend of genome-wide hyper-

| Trained immunity
The classical view of innate versus adaptive immunity is that immunological memory is a unique, hallmark feature of the adaptive response, exemplified by the acquisition of distinct memory states in B cells and T cells, and the ability to generate rapid, antigen-specific responses to subsequent challenge. However, recent studies have supported the concept of "trained immunity," the ability of innate immune cells, such as macrophages and DCs, to adopt adaptive traits, where responses to re-infection/re-challenge are influenced by a previous exposure. 261

| Tissue-resident T cells
Tissue-resident memory T cells (Trm) represent a subset of T cells which occupy tissues and do not recirculate, forming a separate pool distinct from T cells in peripheral blood or secondary lymphoid organs. 269 These cells perform surveillance and rapid effector functions in tissues, at the site of initial antigen recognition. They have been well characterized at mucosal and barrier surfaces, but also F I G U R E 7 "Trained" immunity and consequences of LPS tolerization. In contrast to traditional adaptive response, trained immunity refers to a memory-like response displayed by innate immune cells such as monocytes, macrophages, and DCs. An example is LPS tolerance, a phenomenon characterized by a diminished response upon subsequent LPS challenge. This is thought to have important consequences in sepsis, which can cause death either due to an initial hyperactivation of the immune system and induction of cytokine storm or a delayed immunosuppressive state characterized by secondary infections that is also associated with increased mortality. The suppression of the immune response is in part attributed to epigenetic modifications in the cells, leading to a loss of H3K27ac deposition in important genes, suppressing gene expression [Colour figure can be viewed at wileyonlinelibrary.com]

H3K27ac
"Trained" immunity While interstitial Trm and airway Trm shared a core tissue residency signature including the genes Itgae, Ahr, Cxcr6, and S1p1r, they found an enriched unfolded protein response specifically in airway Trm suggesting an adaptation to the unique environment of the airway. They then turned to bulk ATACseq of these populations to define the epigenetic programmes imbued by the tissue environment, finding a unique chromatin accessibility pattern in airway Trm enriched for binding motifs of Together these findings suggest epigenetic programming is able to balance lineage commitment and plasticity, permitting activation and mobilization of Trm and re-establishment of the Trm phenotype over the time course of an infection.

| Natural killer cells
Natural killer (NK) cells are innate lymphoid cells best known for their cytotoxicity, identifying and killing virally infected or malignantly transformed cells that have downregulated MHC class I. In humans, NK cells consist of two major subsets-CD56 dim CD16 + and CD56 bright CD16 -, the former being the dominant circulating subset that exhibits high cytotoxic capacity. 283  and T-bet, which contribute to rapid induction of effector genes. 291 Similar to Ifng, NKG2D also had a high level of H3K9ac and a low level of H3K4me3, suggestive of active transcription. Lastly, key miRNAs have been identified in the development, maturation, and effector functions of NK cells. 292 These miRNAs play a repressive role in regulating target gene expression, including those encoding perforin (miR-30e, miR-150), granzyme B (miR-378), and IFNγ (miR-146a; via NF-κB signaling).

| Structural cells
While not traditionally considered to play a part in immune responses, increasing evidence supports the conclusion that nonimmune cells that exist alongside specialized immune cell subsets within tissues work in concert with professional immune cells to maintain organ health. For example, in the kidneys, epithelial cells in different anatomical regions show distinct immune functions; pelvic epithelial cells, the first kidney cells encountered by bacteria ascending from the bladder, basally express antimicrobial peptides such as lipocalin 2 (LCN2), an iron chelator that has roles in limiting bacterial growth 293 and is associated with prevention of urinary tract infections, 294 and neutrophil-and MNP-recruiting chemokines, such as CXCL8 and CX3CL1, 26 in homeostasis, suggesting they are primed to respond to infectious challenge. In contrast, podocytes show a different chemokine expression profile, with high expression of CXCL12, 26 with the capacity to attract CXCR3/4-expressing cells, and have been shown to express MHC-II molecules during inflammation. 295 Recently, a multi-omic analysis of endothelial cells, fibroblasts, and epithelial cells across twelve different murine organs revealed organ-specific epigenetic and transcriptomic networks that supported immune activity and homeostasis. 27 The structural cells expressed receptors and ligands enabling interactions with immune cells in an organ-and cell type-specific manner; for example, interactions between endothelial cells with NK cells or monocytes were particularly enriched in kidneys. 27 Specific immune-related genes expressed in kidney endothelium, epithelium, and fibroblasts in homeostasis included Lrp2 (lipoprotein receptor-related protein 2), Spp1 (encoding osteopontin (OPN)), Dysf, and Col4a3 (collagen type IV alpha 3 chain), some of which have disease associations ( Figure 8A); for example, Lrp2 encodes megalin, an endocytic receptor for filtered protein and defects in this protein can cause tubular proteinuria and anti-LRP2 is a model for nephritis called Heymann nephritis 296 and was identified in predicted interactions between kidney endothelium, epithelium, and fibroblasts with monocytes, B cells, and macrophages. 27 Furthermore, OPN receptors include some integrins and CD44, expressed by many immune cells, and OPN-deficient mice demonstrate more severe kidney damage in the nephrotoxic nephritis model of immune complex-mediated glomerulonephritis. 297 The epigenetic landscape of structural cells in this pan-organ study was consistent with the transcriptomic findings, showing open chromatin accessibility profiles in many important genes and transcription factors, including the promoter region of Cdh16 as a kidney-specific effect in the three cell types examined. Importantly, regions around immune-related genes were also observed to be characteristically open in homeostasis, including regions near Tlr9 in the brain and liver, and Stat5a and Stat5b in the heart, intestines, and spleen; high chromatin accessibility for Ifngr1 promoter region was observed in the brain, caecum, spleen, heart, kidneys, and liver 27 ( Figure 8B). This supported the conclusion that the structural cells adopt a primed epigenetic state enabling rapid future immune activation. Indeed, in both lymphocytic choriomeningitis virus (LCMV) infection and following cytokine challenge, the organ structural cells displayed a cell type-and organ-specific activation of immune response genes and pathways. 27 By contrasting the mRNA expression post-challenge with that of the RNA-expression profile and epigenetic profile of cells in homeostasis, a substantial number of the immune genes were found to be initially lowly expressed in homeostasis but were "poised" for immune activation, with higher chromatin accessibility 27 ( Figure 8B). The effects of cytokine stimulation in the kidney were not specifically investigated or functionally validated due to the relatively weak effects of LCMV challenge on structural cell transcriptomes compared to other organs, but it may F I G U R E 8 Epigenetic modification of non-immune cells primes regulation of tissue immunity. A, In the kidney, potential interactions between fibroblasts, endothelial cells, and epithelial cells with monocytes, macrophages, and B cells are predicted from the transcriptome data, with molecules such as Lrp2, Spp1, Bmp7, and Apoe being expressed on the non-immune cells. B, The transcriptomics findings were correlated with increased chromatin accessibility profiles in a cell-type and organ-specific manner. For example, the increased accessibility to the promoter region of renal adhesion molecule Cdh16 was exclusive to kidneys. Importantly, increased chromatin accessibility was observed for regions related to several immune genes including Ifngr1 and Tlr9 during homeostasis. Many immune response genes were "poised" for activation in non-immune cells, characterized as displaying increased chromatin accessibility but low gene expression during homeostasis and high gene expression after challenge. Adapted from Krausgruber et al 27  well be that "primed" gene expression in kidney structural cells may play a role in immune responses to renal injury and infection models.
While this large resource sheds new light on the underappreciated role of "structural immunity," there is likely underlying heterogeneity within cell subtypes that remains to be validated, exemplified by our single-cell characterization of the non-immune landscape in the kidney, 26 and by others across a variety of tissues and organs. Nevertheless, we anticipate future efforts to extend to single-cell resolution, for example, the human cell atlas of fetal transcriptome 139 and epigenome, 298 mapping a more complete understanding of the regulation of immunity.

| CON CLUS I ON S AND FUTURE DIREC TIONS
Epigenetic profiling of tissue immune cells has revealed some of the details of the mechanisms that control the development, recruitment, maintenance, and activation of these cells. The best-studied tissue cell is the macrophage, with detailed knowledge of the epigenetic mechanisms that control their development in embryogenesis, their recruitment and tissue specification, their replenishment from the monocyte pool, and their polarization and activation. As we increasingly appreciate the diversity of immune cells present within organs beyond macrophages, it is evident that much more work is needed to define the mechanisms at play in controlling the transcriptional activity of these cells, along with organ structural cells to generate coordinated, tissue-and stimulus-specific immune responses.
Continuing technological advances should enable the acquisition of this information at single-cell resolution, and their application to human tissue samples in health and disease, including in the kidney, has the potential to identify cell-and tissue-specific epigenetic targets for disease treatments across organs.

ACK N OWLED G EM ENTS
We have no conflicts of interest to declare. ZKT and MRC are sup-

CO N FLI C T O F I NTE R E S T
All authors declare that potential conflicts do not exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.