TREM2 drives microglia response to amyloid-ß via SYK-dependent and -independent pathways.

Genetic studies have highlighted microglia as pivotal in orchestrating Alzheimer's disease (AD). Microglia that adhere to Aß plaques acquire a transcriptional signature, "disease-associated microglia" (DAM), which largely emanates from the TREM2-DAP12 receptor complex that transmits intracellular signals through the protein tyrosine kinase SYK. The human TREM2R47H variant associated with high AD risk fails to activate microglia via SYK. We found that SYK-deficient microglia cannot encase Aß plaques, accelerating brain pathology and behavioral deficits. SYK deficiency impaired the PI3K-AKT-GSK-3ß-mTOR pathway, incapacitating anabolic support required for attaining the DAM profile. However, SYK-deficient microglia proliferated and advanced to an Apoe-expressing prodromal stage of DAM; this pathway relied on the adapter DAP10, which also binds TREM2. Thus, microglial responses to Aß involve non-redundant SYK- and DAP10-pathways. Systemic administration of an antibody against CLEC7A, a receptor that directly activates SYK, rescued microglia activation in mice expressing the TREM2R47H allele, unveiling new options for AD immunotherapy.

The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

Human early-onset dementia caused by DAP12 deficiency reveals a unique signature of dysregulated microglia.

The TREM2-DAP12 receptor complex sustains microglia functions. Heterozygous hypofunctional TREM2 variants impair microglia, accelerating late-onset Alzheimer's disease. Homozygous inactivating variants of TREM2 or TYROBP-encoding DAP12 cause Nasu-Hakola disease (NHD), an early-onset dementia characterized by cerebral atrophy, myelin loss and gliosis. Mechanisms underpinning NHD are unknown. Here, single-nucleus RNA-sequencing analysis of brain specimens from DAP12-deficient NHD individuals revealed a unique microglia signature indicating heightened RUNX1, STAT3 and transforming growth factor-ß signaling pathways that mediate repair responses to injuries. This profile correlated with a wound healing signature in astrocytes and impaired myelination in oligodendrocytes, while pericyte profiles indicated vascular abnormalities. Conversely, single-nuclei signatures in mice lacking DAP12 signaling reflected very mild microglial defects that did not recapitulate NHD. We envision that DAP12 signaling in microglia attenuates wound healing pathways that, if left unchecked, interfere with microglial physiological functions, causing pathology in human. The identification of a dysregulated NHD microglia signature sparks potential therapeutic strategies aimed at resetting microglia signaling pathways.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes.

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr-/- IELs. Loss of IELs in Ahrr-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages.

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-ß pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

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.

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.

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.

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.

Group 1 ILCs: Heterogeneity, plasticity, and transcriptional regulation.

Group 1 innate lymphoid cells (ILCs), comprising ILC1s and natural killer cells (NK cells), belong to a large family of developmentally related innate lymphoid cells that lack rearranged antigen-specific receptors. NK cells and ILC1s both require the transcription factor T-bet for lineage commitment but additionally rely on Eomes and Hobit, respectively, for their development and effector maturation programs. Both ILC1s and NK cells are essential for rapid responses against infections and mediate cancer immunity through production of effector cytokines and cytotoxicity mediators. ILC1s are enriched in tissues and hence generally considered tissue resident cells whereas NK cells are often considered circulatory. Despite being deemed different cell types, ILC1s and NK cells share many common features both phenotypically and functionally. Recent studies employing single cell RNA sequencing (scRNA-seq) technology have exposed previously unappreciated heterogeneity in group 1 ILCs and further broaden our understanding of these cells. Findings from these studies imply that ILC1s in different tissues and organs share a common signature but exhibit some unique characteristics, possibly stemming from tissue imprinting. Also, data from recent fate mapping studies employing Hobit, RORγt, and polychromic reporter mice have greatly advanced our understanding of the developmental and effector maturation programs of these cells. In this review, we aim to outline the fundamental traits of mouse group 1 ILCs and explore recent discoveries related to their developmental programs, phenotypic heterogeneity, plasticity, and transcriptional regulation.

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.

The Tumor Necrosis Factor Superfamily Member RANKL Suppresses Effector Cytokine Production in Group 3 Innate Lymphoid Cells.

While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity.

NKp46+ ILC3s promote early neutrophil defense against Clostridioides difficile infection through GM-CSF secretion.

Clostridioides difficile infection (CDI) is a common cause of antibiotic-associated colitis. C. difficile proliferates and produces toxins that damage the colonic epithelium, leading to symptoms ranging from mild diarrhea to severe pseudomembranous colitis. The host's innate response to CDI occurs in two phases: an early phase in which neutrophils reduce the bacterial load and a late phase involving repair mechanisms to restore epithelial integrity. Group 3 innate lymphoid cells (ILC3s) are crucial in protecting the gut from CDI. Previous studies have shown that ILC3-derived IL-22 is essential in the late phase of CDI for epithelial repair and maintaining an intestinal microbiota that competes with C. difficile, preventing its expansion. Our study finds that ILC3s also protect during the early stages of CDI by sustaining neutrophils through GM-CSF. Less neutrophil production, accumulation, and activation was evident in ILC3-deficient mice than in wild-type (WT) mice, which led to exacerbated symptoms, impaired pathogen clearance, a compromised epithelial barrier, and increased mortality. The adoptive transfer of ILC3s into ILC3-deficient mice restored neutrophil responses and improved disease outcomes. Both in vitro and in vivo experiments revealed that GM-CSF production by ILC3s is crucial for neutrophil production and effective resistance during CDI. Using mice lacking NKp46+ ILC3s, we found that this subset significantly contributes to GM-CSF production in CDI. These findings highlight the critical role of the ILC3-neutrophil connection in early innate responses to CDI. Enhancing ILC3 production of GM-CSF could be a promising strategy for improving host defense against CDI and other enteric infections.

Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions.

CD96, CD226 (DNAM-1) and TIGIT belong to an emerging family of receptors that interact with nectin and nectin-like proteins. CD226 activates natural killer (NK) cell-mediated cytotoxicity, whereas TIGIT reportedly counterbalances CD226. In contrast, the role of CD96, which shares the ligand CD155 with CD226 and TIGIT, has remained unclear. In this study we found that CD96 competed with CD226 for CD155 binding and limited NK cell function by direct inhibition. As a result, Cd96(-/-) mice displayed hyperinflammatory responses to the bacterial product lipopolysaccharide (LPS) and resistance to carcinogenesis and experimental lung metastases. Our data provide the first description, to our knowledge, of the ability of CD96 to negatively control cytokine responses by NK cells. Blocking CD96 may have applications in pathologies in which NK cells are important.

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.

SLC7A8 is a key amino acids supplier for the metabolic programs that sustain homeostasis and activation of type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2) are innate counterparts of T helper 2 (Th2) cells that maintain tissue homeostasis and respond to injuries through rapid interleukin (IL)-5 and IL-13 secretion. ILC2s depend on availability of arginine and branched-chain amino acids for sustaining cellular fitness, proliferation, and cytokine secretion in both steady state and upon activation. However, the contribution of amino acid transporters to ILC2 functions is not known. Here, we found that ILC2s selectively express Slc7a8, encoding a transporter for arginine and large amino acids. Slc7a8 was expressed in ILC2s in a tissue-specific manner in steady state and was further increased upon activation. Genetic ablation of Slc7a8 in lymphocytes reduced the frequency of ILC2s, suppressed IL-5 and IL-13 production upon stimulation, and impaired type 2 immune responses to helminth infection. Consistent with this, Slc7a8-deficient ILC2s also failed to induce cytokine production and recruit eosinophils in a model of allergic lung inflammation. Mechanistically, reduced amino acid availability due to Slc7a8 deficiency led to compromised mitochondrial oxidative phosphorylation, as well as impaired activation of mammalian target of rapamycin and c-Myc signaling pathways. These findings identify Slc7a8 as a key supplier of amino acids for the metabolic programs underpinning fitness and activation of ILC2s.