Genetics and environment distinctively shape the human immune cell epigenome.

The epigenomic landscape of human immune cells is dynamically shaped by both genetic factors and environmental exposures. However, the relative contributions of these elements are still not fully understood. In this study, we employed single-nucleus methylation sequencing and ATAC-seq to systematically explore how pathogen and chemical exposures, along with genetic variation, influence the immune cell epigenome. We identified distinct exposure-associated differentially methylated regions (eDMRs) corresponding to each exposure, revealing how environmental factors remodel the methylome, alter immune cell states, and affect transcription factor binding. Furthermore, we observed a significant correlation between changes in DNA methylation and chromatin accessibility, underscoring the coordinated response of the epigenome. We also uncovered genotype-associated DMRs (gDMRs), demonstrating that while eDMRs are enriched in regulatory regions, gDMRs are preferentially located in gene body marks, suggesting that exposures and genetic factors exert differential regulatory control. Notably, disease-associated SNPs were frequently colocalized with meQTLs, providing new cell-type-specific insights into the genetic basis of disease. Our findings underscore the intricate interplay between genetic and environmental factors in sculpting the immune cell epigenome, offering a deeper understanding of how immune cell function is regulated in health and disease.

The major bottleneck of understanding human immunology and immunopathology is the lack of relevant animal models. A robust small animal model for hypothesis testing and mechanistic research on human immunology is needed and should meet the following criteria. First, it has well-studied hematolymphoid organs and target cells which are human. Second, human immune pathology including infection diseases can be modeled in vivo. Third, it is genetically inbred and can be manipulated via immunological, genetic and pharmacological means. The immunodeficient mouse transplanted with human immune organs and cells is the obvious candidate. A number of human–mouse chimeric models were developed in the late 1980s, but with only limited success, due to a lack of significant human immune responses in the SCID-hu Thy/Liv mouse1, 2 or selective engraftment of xeno-reactive human T cells in the hu-PBL-SCID mouse.3, 4

We recently found that γ-aminobutyric acid (GABA) protects mouse islet β cells. It prevented autoimmune type 1 diabetes in mice, induced islet β-cell regeneration, and exerted immunoinhibitory effects. However, it is not known whether GABA would be equally active on human islet and immune cells. In vitro culture of human islets and immune cells with or without GABA and immunosuppressive drugs. In vitro analysis of apoptosis, proliferation, nuclear factor (NF)-κB activation, calcium signaling, and insulin secretion. GABA reduced human islet cell apoptosis in culture, such that the yield of live cells was approximately tripled after 1 week, and it stimulated insulin secretion. It protected against the deleterious effects of rapamycin, tacrolimus, and mycophenolate mofetil. In human immune cells, GABA had inhibitory effects similar to mouse cells, such as suppressed anti-CD3-stimulated T-cell proliferation, in a GABA type A receptor-dependent fashion. The immunosuppressive mechanisms have been unclear, but we found that GABA blocked calcium influx, which is a key activation signal. GABA also suppressed NF-κB activation in both human islet cells and immune cells. We found that it could be combined with rapamycin to increase its suppressive effects. GABA improved human islet cell survival and had suppressive effects on human immune cells. It inhibited canonical NF-κB activation in both islet and immune cells. This is important because activation of this pathway is detrimental to islet cells and likely promotes damaging autoimmunity and alloreactivity against transplanted islets. These findings suggest that GABA might find applications in clinical islet transplantation.

Tributyltin (TBT) is an environmental contaminant that has been used for several industrial, agricultural and household purposes. Despite limited bans on the usage of Tributyltin, the stability and toxicity of the compound may prove to have negative effects on immune function in terrestrial mammals. Studies show that there is an increase in the incidences of tumors and decreased immune cell function in TBT-exposed mammals. TBT interferes with the ability of human natural killer (NK) cells to lyse target cells; it also alters the secretion of the pro-inflammatory cytokines Interferon gamma (IFNγ) and Tumor necrosis factor alpha (TNFα) from human immune cells ex vivo. Any dysregulation of the cytokine communication network may have detrimental effects on immune function, as cytokines play a key role in the regulation of immune responsiveness. There have been no in vivo studies on the effects of exposure to TBT on the cytokine communication network. The effects of 24 hour exposure to TBT on the production of IFNγ and TNFα from human immune cells were analyzed in an ex vivo system using western blotting. The serum of TBT-exposed mice was analyzed for changes in the levels of cytokine secretion and production using a Milliplex mouse cytokine/chemokine magnetic bead premixed 32 plex kit and western blotting; the effects of TBT-exposure was assessed using a time curve of 6h, 12h, 24h, 48h and control. TBT increased the levels of the cytokines IFNγ, TNFα, IL1β, IL5, IL7, IL12βp40, IL13, IL15, MIP1β, MIP2 and RANTES in the serum at a minimum of one time point. IFNγ and TNFα secretion and production from human cells also increased, showing striking agreement in the response to TBT between the human and mouse systems. Supported by: NIH grant 5U54CA163066-03 Citation Format: Shanieek T. Lawrence, Margaret Whalen, Samuel Pellom, Anil Shanker. Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2015-2735

Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells

Genetics of congenital heart defects: is it not all in the DNA?

Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle we supplemented NSG/NOG mice with human hematopoietic stem cells (HSC) and subsequently examined growth characteristics of the non-small cell lung cancer (NSCLC) PDX model LXFA 923 in these mice. In parallel we monitored the presence of human and murine immune cells in different organs of the mouse. HSC (2×106) cells were isolated from healthy donors and injected intravenously into sub-lethally irradiated NSG or NOG mice (n = 43 mice in 3 ind. exp.). After 8 weeks LXFA 923 was transplanted subcutaneously (s.c.) into the pretreated mice. Murine peripheral blood was examined by flow cytometry for common murine and human markers expressed on immune cells (hCD14, mCD14, hCD3, mCD3, hCD56, mCD56, hCD19) once weekly. At the end of the experiment tumors and organs were analyzed for human cancer (CD44, CD133, CDCP1, CD166, CD24) and immune cell markers (hCD14, hCD3, hCD56, hCD19) by flow cytometry. Tumors and organs were additionally histologically and immunohistochemically examined. Growth of the implanted tumors was monitored by caliper measurement. Mice bearing only the subcutaneous PDX or the HSC served as control groups. Stable engraftment of human immune cells in immune-compromized mice was successfully achieved. Human immune cells expressing T-, B-, NK- and stem cell markers could be detected in different compartments (bone marrow, peripheral blood and spleen) of the tumor-bearing as well as non-tumor bearing mice. Furthermore, infiltrates of human monocytes (CD14+) as well as T cells (CD3+) could be detected in s.c. implanted tumor tissue. Implantation of LXFA 923 did not influence the proliferation of human immune cells in recipient mice. Growth behavior of the s.c. implanted PDX was not affected by the engraftment of HSC in the murine host. The histological architecture of LXFA 923 was similar when implanted s.c. in humanized or immunodeficient mice and it still closely resembles the patient donor material. In conclusion, our investigations validate the analysis of PDX in mice engrafted with human immune cells, as it enables the interaction of tumor cells with human immune cells as well as with murine stroma to be investigated. This preclinical PDX based in vivo platform provides a further step to support the development of new drugs targeting the host immune response. Citation Format: Eva Oswald, Kerstin Klingner, Dorothee Lenhard, Gabriele Niedermann, Julia B. Schüler. NSCLC PDX model for the evaluation of immuno-oncological treatment strategies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2015-5023

The liver is an immunological organ with a distinct immune cell profile. Although the composition and function of liver immune cells have been widely investigated, the mechanisms regulating the development and homeostasis of the specialized immune system, especially in humans, remain largely unknown. Herein, we address this question in humanized mice (hu-mice) that were constructed by transplantation of human fetal thymus and CD34+ hematopoietic stem/progenitor cells in immunodeficient mice with or without autologous human hepatocyte engraftment. Although the levels of human immune cell reconstitution in peripheral blood and spleen were comparable between hu-mice with and without human hepatocyte engraftment, the former group showed that human immune cell reconstitution in the liver was significantly improved. Notably, human immune cells, including Kupffer cells, dendritic cells and natural killer cells, were shown to be closely colocalized with human hepatocytes in the liver. Human hepatocytes engrafted in the mouse liver were found to produce IL-3, IL-15, GM-CSF, M-CSF, MCP-1, CXCL-1 and CXCL-10, which are known to be important for immune cell development, differentiation, tissue migration and retention, and have no or poor cross-reaction between humans and mice. Furthermore, human hepatocytes were able to support human immune cell survival and expansion in an in vitro co-culture assay. This study demonstrates an essential role for hepatocytes in the development and maintenance of the liver immune cell profile. The hu-mouse model with human autologous immune cell and hepatocyte reconstitution has potential for use in studies of the pathogenesis of liver immune disorders such as hepatotropic virus infections.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Beta-1,3/1,6 glucans (BGs) are a diverse class of PAMPs. Structurally, BGs can vary on the basis of chain length, types of linkages, branching, and tertiary structure. BGs have been reported to activate various types of innate immune cells; most studies demonstrate the use of particulate BGs activating murine immune cells. Unlike particulate BGs, most soluble BGs demonstrated minimal activities typically associated with innate immune cell activation, such as induction of cytokines. Hence, little is known regarding the types of human immune cells or receptors that engage soluble BGs, and if structurally distinct BGs differ in their cell selectivity or receptor requirements. The objective of this study was to determine the types of human immune cells that soluble BGs bind to, and to determine the receptors required for binding. Since BGs have been implicated in interacting with Dectin-1 and CR3 expressing cells, we hypothesize that human innate immune cells that express these receptors will bind soluble BGs. Specific binding of BGs was evaluated by flow cytometry on primary human immune cells and cell lines using BG specific antibody and DTAF-labeled BGs. In addition, these studies were conducted using three structurally distinct, purified, and chemically characterized BGs. Key findings demonstrate that on cells that express both CR3 and Dectin-1, CR3 is a critical receptor for binding soluble BGs and not all BGs bind equivalently to human innate immune cells.

Somatostatin is a neuropeptide that is widely distributed throughout the body. It was first identified as a growth hormone release‐inhibiting factor synthesized in the hypothalamus. Outside the central nervous system (CNS), the peptide is present in endocrine as well as non‐endocrine tissues. Somatostatin functions as a peptide with a generally inhibitory action in the CNS and endocrine system. In the CNS, it can act as a neurotransmitter, while in peripheral tissues, it regulates endocrine and exocrine secretion and acts as a modulator of motor activity in the gastrointestinal tract. Besides these actions, somatostatin has also been shown to have antiproliferative effects in vitro. Somatostatin binds to five different subtype receptors (sst) which are differently expressed by various tissues. These receptors have been described also in the immune system of various species including humans. It is therefore a long known fact that human immune cells and their progenitors can express somatostatin receptors. As a consequence, ssts were described in primary and secondary human immune organs. Somatostatin produced by immune cells may act as an autocrine or paracrine regulator within the local immune microenvironment in mice. The synthesis, however, of somatostatin has not been demonstrated in human immune cells. In a recent study, the expression of another somatostatin‐like peptide; cortistatin‐17 (CST) was found in human lymphoid tissues, immune cells and lymphoid cell lines. On the basis of these observations, a role for CST as an endogenous ligand for sst in the human immune system, rather than SS itself was hypothesized. Somatostatin receptor expression can be detected in vivo somatostatin receptor scintigraphy after injection of 111In‐labelled octreotide, a somatostatin analogue. This technique is used extensively for the localization of neuroendocrine tumours and other malignancies that express high levels of sst. Among the non‐neuroendocrine tumours that can be visualized by octreotide scintigraphy are malignant lymphomas, both T and B non‐Hodgkin's lymphomas and Hodgkin's disease lymphomas. In a number of infectious diseases (e.g. tuberculosis), autoimmune diseases (e.g. Graves' ophthalmopathy) and other immune‐mediated diseases (e.g. sarcoidosis and rheumatoid arthritis), the sites of inflammation can also be visualized. Based on the receptor pattern in autoimmune diseases, controlled studies are warranted to investigate the efficacy of somatostatin analogues in the treatment of autoimmune diseases like rheumatoid arthritis. In rheumatoid arthritis, not only immune cells can be targeted by these analogues but also synoviocytes and synovial blood vessels. Moreover, receptor expression during treatment in such diseases can be monitored by octreotide scintigraphy.

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.

Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells by autoreactive T lymphocytes, leading to insulin deficiency and lifelong insulin dependence. It develops in genetically predisposed individuals, triggered by environmental or immunological factors. Although the exact causes of T1D remain unknown, the autoimmune pathogenesis of the disease is clearly indicated by the genetic risk conferred by allelic human leukocyte antigens (HLA), the almost obligatory presence of islet cell autoantibodies (AAbs) and immune cell infiltration of pancreatic islets from patients. At the same time, epidemiological data point to a role of environmental factors, notably enteroviral infections, in the disease, although precise causative links between specific pathogens and T1D have been difficult to establish. Studies of human pancreas organs from patients made available through repositories and the advent of high‐dimensional high‐throughput technologies for genomic and proteomic studies have significantly elucidated our understanding of the disease in recent years and provided mechanistic insights that can be exploited for innovative targeted therapeutic approaches. This short overview will summarise current salient knowledge on immune cell and beta cell dysfunction in T1D pathogenesis.Plain Language SummaryType 1 diabetes (T1D) is a chronic disease where the body's own immune system attacks and destroys the insulin‐producing beta cells in the pancreas. This leads to a lack of insulin, a hormone essential for regulating blood sugar, which means people with T1D need insulin for life. The disease can develop at any age but is most diagnosed in children and young adults. Despite advances in treatment, T1D still significantly reduces life expectancy, especially in countries with fewer healthcare resources. T1D develops in people with a genetic predisposition, often triggered by environmental factors such as viral infections or changes in the gut microbiome. The disease progresses silently through three stages: Stage 1: Autoantibodies to beta cell components appear, signalling the immune system is reacting against the pancreas, but there are no symptoms; Stage 2: Beta cell function starts to decline, but fasting blood sugar is still normal; Stage 3: Enough beta cells are destroyed that fasting blood sugar rises, and symptoms of diabetes appear. The risk of progressing from stage 1 to full‐blown diabetes is about 35–50% within five years, and even higher from stage 2. Over 60 genes are linked to T1D risk, most of which affect how the immune system works. The strongest genetic risk comes from specific versions of histocompatibility genes, which help the immune system distinguish between the body's own cells and invaders. Some types of these genes make it easier for the immune system to mistakenly attack beta cells. However, 90% of people diagnosed with T1D have no family member with T1D, showing that genetics is only part of the story. Environmental factors also play a big role. For example, certain viral infections, especially with viruses infecting the intestine, are associated with a higher risk of developing T1D. The gut microbiome ‐ the community of bacteria living in our intestines ‐ also influences risk, with healthier, more diverse microbiomes appearing to offer some protection. In T1D, immune cells ‐ especially so‐called T lymphocytes – mistake beta cells in the pancreas for threats and destroy them. This process is called autoimmunity. The attack is often reflected by the presence of autoantibodies against proteins found in beta cells. Over time, as more beta cells are lost, the body can no longer produce enough insulin, leading to the symptoms of diabetes. Interestingly, not all people with T1D have the same pattern of disease. For example, children diagnosed before age 7 often have more aggressive disease, more autoantibodies, and stronger genetic risk factors than those diagnosed later. Much of our understanding of T1D has come from studying animal models, but new technologies now allow researchers to study human pancreas tissue and blood immune cells in greater detail. Scientists are also exploring how the gut microbiome, diet, and environmental exposures contribute to T1D risk and progression. Treatment currently focuses on replacing insulin, but researchers are working on therapies that target the immune system or aim to protect or replace beta cells. Strategies include immunotherapy, gene therapy, and even modifying the gut microbiome. The goal is to prevent or reverse the disease, not just manage its symptoms. In summary, T1D is a complex autoimmune disease influenced by both genes and the environment. It progresses silently before symptoms appear, and while insulin therapy is life‐saving, new research is paving the way for treatments that could one day halt or even prevent the disease.

The plague agent, Yersinia pestis, employs a type III secretion system (T3SS) to selectively destroy human immune cells, thereby enabling its replication in the bloodstream and transmission to new hosts via fleabite. The host factors responsible for the selective destruction of immune cells by plague bacteria were not known. Here we show that LcrV, the needle cap protein of the Y. pestis T3SS, binds N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality, however N-formylpeptide receptor deficient animals exhibit increased survival and plague-protective antibody responses. We identified FPR1 p.R190W as a candidate human resistance allele that protects neutrophils from Y. pestis T3SS. These findings reveal the plague receptor on immune cells and show that FPR1 mutations provide for plague survival, which appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.

The mechanisms how environmental compounds influence the human immune system are unknown. The environmentally sensitive transcription factor aryl hydrocarbon receptor (AHR) has immune-modulating functions and responds to small molecules. Cytochrome P4501 enzymes (CYP1) act downstream of the AHR and metabolize small molecules. However, it is currently unknown whether CYP1 activity is relevant for immune modulation. We studied the interdependence of CYP1 and AHR in human primary immune cells using pharmacological methods. CYP1 inhibition increased the expression levels of the stem cell factor receptor (c-Kit) and interleukin (IL)-22 but decreased IL-17. Single cell analyses showed that CYP1 inhibition especially promoted CD4+ helper T (Th) cells that co-express c-Kit and IL-22 simultaneously. The addition of an AHR antagonist reversed all these effects. In addition to T cells, we screened other human immune cells for CYP and found cell-specific fingerprints, suggesting that similar mechanisms are present in multiple immune cells. We describe a feedback loop yet unknown in human immune cells where CYP1 inhibition resulted in an altered AHR-dependent immune response. This mechanism relates CYP1-dependent metabolism of environmental small molecules to human immunity.