Association of Somatic TET2 Mutations With Giant Cell Arteritis.

OBJECTIVE: Giant cell arteritis (GCA) is an age-related vasculitis. Prior studies have identified an association between GCA and hematologic malignancies (HMs). How the presence of somatic mutations that drive the development of HMs, or clonal hematopoiesis (CH), may influence clinical outcomes in GCA is not well understood. METHODS: To examine an association between CH and GCA, we analyzed sequenced exomes of 470,960 UK Biobank (UKB) participants for the presence of CH and used multivariable Cox regression. To examine the clinical phenotype of GCA in patients with and without somatic mutations across the spectrum of CH to HM, we performed targeted sequencing of blood samples and electronic health record review on 114 patients with GCA seen at our institution. We then examined associations between specific clonal mutations and GCA disease manifestations. RESULTS: UKB participants with CH had a 1.48-fold increased risk of incident GCA compared to UKB participants without CH. GCA risk was highest among individuals with cytopenia (hazard ratio [HR] 2.98, P = 0.00178) and with TET2 mutation (HR 2.02, P = 0.00116). Mutations were detected in 27.2% of our institutional GCA cohort, three of whom had HM at GCA diagnosis. TET2 mutations were associated with vision loss in patients with GCA (odds ratio 4.33, P = 0.047). CONCLUSIONS: CH increases risk for development of GCA in a genotype-specific manner, with the greatest risk being conferred by the presence of mutations in TET2. Somatic TET2 mutations likewise increase the risk of GCA-associated vision loss. Integration of somatic genetic testing in GCA diagnostics may be warranted in the future.

Clonal haematopoiesis and dysregulation of the immune system.

Age-related diseases are frequently linked to pathological immune dysfunction, including excessive inflammation, autoreactivity and immunodeficiency. Recent analyses of human genetic data have revealed that somatic mutations and mosaic chromosomal alterations in blood cells - a condition known as clonal haematopoiesis (CH) - are associated with ageing and pathological immune dysfunction. Indeed, large-scale epidemiological studies and experimental mouse models have demonstrated that CH can promote cardiovascular disease, chronic obstructive pulmonary disease, chronic liver disease, osteoporosis and gout. The genes most frequently mutated in CH, the epigenetic regulators TET2 and DNMT3A, implicate increased chemokine expression and inflammasome hyperactivation in myeloid cells as a possible mechanistic connection between CH and age-related diseases. In addition, TET2 and DNMT3A mutations in lymphoid cells have been shown to drive methylation-dependent alterations in differentiation and function. Here we review the observational and mechanistic studies describing the connection between CH and pathological immune dysfunction, the effects of CH-associated genetic alterations on the function of myeloid and lymphoid cells, and the clinical and therapeutic implications of CH as a target for immunomodulation.

The Immunopathology of Giant Cell Arteritis Across Disease Spectra.

Giant cell arteritis (GCA) is a granulomatous systemic vasculitis of large- and medium-sized arteries that affects the elderly. In recent years, advances in diagnostic imaging have revealed a greater degree of large vessel involvement than previously recognized, distinguishing classical cranial- from large vessel (LV)- GCA. GCA often co-occurs with the poorly understood inflammatory arthritis/bursitis condition polymyalgia rheumatica (PMR) and has overlapping features with other non-infectious granulomatous vasculitides that affect the aorta, namely Takayasu Arteritis (TAK) and the more recently described clinically isolated aortitis (CIA). Here, we review the literature focused on the immunopathology of GCA on the background of the three settings in which comparisons are informative: LV and cranial variants of GCA; PMR and GCA; the three granulomatous vasculitides (GCA, TAK, and CIA). We discuss overlapping and unique features between these conditions across clinical presentation, epidemiology, imaging, and conventional histology. We propose a model of GCA where abnormally activated circulating cells, especially monocytes and CD4+ T cells, enter arteries after an unknown stimulus and cooperate to destroy it and review the evidence for how this mechanistically occurs in active disease and improves with treatment.

Circadian rhythm-dependent and circadian rhythm-independent impacts of the molecular clock on type 3 innate lymphoid cells.

Many gut functions are attuned to circadian rhythm. Intestinal group 3 innate lymphoid cells (ILC3s) include NKp46+ and NKp46- subsets, which are RORγt dependent and provide mucosal defense through secretion of interleukin-22 (IL-22) and IL-17. Because ILC3s highly express some key circadian clock genes, we investigated whether ILC3s are also attuned to circadian rhythm. We noted circadian oscillations in the expression of clock and cytokine genes, such as REV-ERBα, IL-22, and IL-17, whereas acute disruption of the circadian rhythm affected cytokine secretion by ILC3s. Because of prominent and rhythmic expression of REV-ERBα in ILC3s, we also investigated the impact of constitutive deletion of REV-ERBα, which has been previously shown to inhibit the expression of a RORγt repressor, NFIL3, while also directly antagonizing DNA binding of RORγt. Development of the NKp46+ ILC3 subset was markedly impaired, with reduced cell numbers, RORγt expression, and IL-22 production in REV-ERBα-deficient mice. The NKp46- ILC3 subsets developed normally, potentially due to compensatory expression of other clock genes, but IL-17 secretion paradoxically increased, probably because RORγt was not antagonized by REV-ERBα. We conclude that ILC3s are attuned to circadian rhythm, but clock regulator REV-ERBα also has circadian-independent impacts on ILC3 development and functions due to its roles in the regulation of RORγt.

Publisher Correction: Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.

Circadian clock protein Rev-erbα regulates neuroinflammation.

Circadian dysfunction is a common attribute of many neurodegenerative diseases, most of which are associated with neuroinflammation. Circadian rhythm dysfunction has been associated with inflammation in the periphery, but the role of the core clock in neuroinflammation remains poorly understood. Here we demonstrate that Rev-erbα, a nuclear receptor and circadian clock component, is a mediator of microglial activation and neuroinflammation. We observed time-of-day oscillation in microglial immunoreactivity in the hippocampus, which was disrupted in Rev-erbα-/- mice. Rev-erbα deletion caused spontaneous microglial activation in the hippocampus and increased expression of proinflammatory transcripts, as well as secondary astrogliosis. Transcriptomic analysis of hippocampus from Rev-erbα-/- mice revealed a predominant inflammatory phenotype and suggested dysregulated NF-κB signaling. Primary Rev-erbα-/- microglia exhibited proinflammatory phenotypes and increased basal NF-κB activation. Chromatin immunoprecipitation revealed that Rev-erbα physically interacts with the promoter regions of several NF-κB-related genes in primary microglia. Loss of Rev-erbα in primary astrocytes had no effect on basal activation but did potentiate the inflammatory response to lipopolysaccharide (LPS). In vivo, Rev-erbα-/- mice exhibited enhanced hippocampal neuroinflammatory responses to peripheral LPS injection, while pharmacologic activation of Rev-erbs with the small molecule agonist SR9009 suppressed LPS-induced hippocampal neuroinflammation. Rev-erbα deletion influenced neuronal health, as conditioned media from Rev-erbα-deficient primary glial cultures exacerbated oxidative damage in cultured neurons. Rev-erbα-/- mice also exhibited significantly altered cortical resting-state functional connectivity, similar to that observed in neurodegenerative models. Our results reveal Rev-erbα as a pharmacologically accessible link between the circadian clock and neuroinflammation.

Two Distinct Myeloid Subsets at the Term Human Fetal-Maternal Interface.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1ß and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-ß signaling.

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-ß family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-ß family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-ß signaling mediated by the cytokine receptor TGFßR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-ß. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-ß signaling in NK cells.

IL-15 sustains IL-7R-independent ILC2 and ILC3 development.

The signals that maintain tissue-resident innate lymphoid cells (ILC) in different microenvironments are incompletely understood. Here we show that IL-7 receptor (IL-7R) is not strictly required for the development of any ILC subset, as residual cells persist in the small intestinal lamina propria (siLP) of adult and neonatal Il7ra-/- mice. Il7ra-/- ILC2 primarily express an ST2- phenotype, but are not inflammatory ILC2. CCR6+ ILC3, which express higher Bcl-2 than other ILC3, are the most abundant subset in Il7ra-/- siLP. All ILC subsets are functionally competent in vitro, and are sufficient to provide enhanced protection to infection with C. rodentium. IL-15 equally sustains wild-type and Il7ra-/- ILC survival in vitro and compensates for IL-7R deficiency, as residual ILCs are depleted in mice lacking both molecules. Collectively, these data demonstrate that siLP ILCs are not completely IL-7R dependent, but can persist partially through IL-15 signalling.

Immune modules shared by innate lymphoid cells and T cells.

In recent years, innate lymphoid cells (ILCs) have emerged as innate correlates to T cells. The similarities between ILCs and T cells indicate that lymphocytes of fundamentally distinct lineages can share core "immune modules" that encompass transcriptional circuitry and effector functions while using nonredundant complementary mechanisms of pattern recognition to enact these functions. We review modules currently recognized to be shared between ILCs and T cells.

Transforming Growth Factor-ß Signaling Guides the Differentiation of Innate Lymphoid Cells in Salivary Glands.

The signals guiding differentiation of innate lymphoid cells (ILCs) within tissues are not well understood. Salivary gland (SG) ILCs as well as liver and intestinal intraepithelial ILC1 have markers that denote tissue residency and transforming growth factor-ß (TGF-ß) imprinting. We deleted Tgfbr2 in cells expressing the ILC and NK marker NKp46 and found that SG ILCs were reduced in number. They lost distinct tissue markers, such as CD49a, and the effector molecules TRAIL and CD73. Expression of the transcription factor Eomes, which promotes NK cell differentiation, was elevated. Conversely, Eomes deletion in NKp46(+) cells enhanced TGF-ß-imprinting of SG ILCs. Thus, TGF-ß induces SG ILC differentiation by suppressing Eomes. TGF-ß acted through a JNK-dependent, Smad4-independent pathway. Transcriptome analysis demonstrated that SG ILCs had characteristic of both NK cells and ILC1. Finally, TGF-ß imprinting of SG ILCs was synchronized with SG development, highlighting the impact of tissue microenvironment on ILC development.

Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets.

Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation.

Group 3 ILCs (ILC3s) are innate sources of IL-22 and IL-17 and include lymphoid tissue-inducer (LTi)-like and NKp46(+) subsets. Both depend on RORγt and aryl hydrocarbon receptor, but NKp46(+)ILC3s also require Notch and T-bet for their development and are transcriptionally distinct. The extent to which these subsets have unique functions, especially in the context of T cell- and B cell-sufficient mice, remains largely unclear. To investigate the specific function of NKp46(+)ILC3s among other ILC3 subsets and T cells, we generated mice selectively lacking NKp46(+)ILC3s or all ILC3s and crossed them to T cell-deficient mice, thus maintaining B cells in all mice. In mice lacking T cells, NKp46(+)ILC3s were sufficient to promote inflammatory monocyte accumulation in the anti-CD40 innate colitis model through marked production of GM-CSF. In T cell-competent mice, lack of NKp46(+)ILCs had no impact on control of intestinal C. rodentium infection, whereas lack of all ILC3s partially impaired bacterial control. Thus, NKp46(+)ILC3s have a unique capacity to promote inflammation through GM-CSF-induced accumulation of inflammatory monocytes, but are superseded by LTi-like ILC3s and T cells in controlling intestinal bacterial infection.

TREM2 sustains microglial expansion during aging and response to demyelination.

Microglia contribute to development, homeostasis, and immunity of the CNS. Like other tissue-resident macrophage populations, microglia express the surface receptor triggering receptor expressed on myeloid cells 2 (TREM2), which binds polyanions, such as dextran sulphate and bacterial LPS, and activates downstream signaling cascades through the adapter DAP12. Individuals homozygous for inactivating mutations in TREM2 exhibit demyelination of subcortical white matter and a lethal early onset dementia known as Nasu-Hakola disease. How TREM2 deficiency mediates demyelination and disease is unknown. Here, we addressed the basis for this genetic association using Trem2(-/-) mice. In WT mice, microglia expanded in the corpus callosum with age, whereas aged Trem2(-/-) mice had fewer microglia with an abnormal morphology. In the cuprizone model of oligodendrocyte degeneration and demyelination, Trem2(-/-) microglia failed to amplify transcripts indicative of activation, phagocytosis, and lipid catabolism in response to myelin damage. As a result, Trem2(-/-) mice exhibited impaired myelin debris clearance, axonal dystrophy, oligodendrocyte reduction, and persistent demyelination after prolonged cuprizone treatment. Moreover, myelin-associated lipids robustly triggered TREM2 signaling in vitro, suggesting that TREM2 may directly sense lipid components exposed during myelin damage. We conclude that TREM2 is required for promoting microglial expansion during aging and microglial response to insults of the white matter.

TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial surface receptor that triggers intracellular protein tyrosine phosphorylation. Recent genome-wide association studies have shown that a rare R47H mutation of TREM2 correlates with a substantial increase in the risk of developing Alzheimer's disease (AD). To address the basis for this genetic association, we studied TREM2 deficiency in the 5XFAD mouse model of AD. We found that TREM2 deficiency and haploinsufficiency augment ß-amyloid (Aß) accumulation due to a dysfunctional response of microglia, which fail to cluster around Aß plaques and become apoptotic. We further demonstrate that TREM2 senses a broad array of anionic and zwitterionic lipids known to associate with fibrillar Aß in lipid membranes and to be exposed on the surface of damaged neurons. Remarkably, the R47H mutation impairs TREM2 detection of lipid ligands. Thus, TREM2 detects damage-associated lipid patterns associated with neurodegeneration, sustaining the microglial response to Aß accumulation.

Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets.

The recognized diversity of innate lymphoid cells (ILCs) is rapidly expanding. Three ILC classes have emerged, ILC1, ILC2 and ILC3, with ILC1 and ILC3 including several subsets. The classification of some subsets is unclear, and it remains controversial whether natural killer (NK) cells and ILC1 cells are distinct cell types. To address these issues, we analyzed gene expression in ILCs and NK cells from mouse small intestine, spleen and liver, as part of the Immunological Genome Project. The results showed unique gene-expression patterns for some ILCs and overlapping patterns for ILC1 cells and NK cells, whereas other ILC subsets remained indistinguishable. We identified a transcriptional program shared by small intestine ILCs and a core ILC signature. We revealed and discuss transcripts that suggest previously unknown functions and developmental paths for ILCs.

Innate lymphoid cells: new insights into function and development.

Here, we illustrate the complexity of ILC subsets and discuss novel functions, focusing on emerging mechanisms of crosstalk with other immune cells and the microbiota. Furthermore, we highlight recent insights into the development of ILCs, including the common pathways they share as well as points of divergence between ILC groups and subsets.

GI motility: microbiota and macrophages join forces.

Gastrointestinal motility causes movement of food during digestion through contractions of the gut smooth muscle. The enteric nervous system regulates these events, and Muller et al. now find that its interaction with the immune system, in concert with gut microbiota, provides an additional layer of regulation to this complex task.