Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Identities from Microglia.

Microglia are thought to originate exclusively from primitive macrophage progenitors in the yolk sac (YS) and to persist throughout life without much contribution from definitive hematopoiesis. Here, using lineage tracing, pharmacological manipulation, and RNA-sequencing, we elucidated the presence and characteristics of monocyte-derived macrophages (MDMs) in the brain parenchyma at baseline and during microglia repopulation, and defined the core transcriptional signatures of brain-engrafted MDMs. Lineage tracing mouse models revealed that MDMs transiently express CD206 during brain engraftment as CD206 + microglia precursors in the YS. We found that brain-engrafted MDMs exhibit transcriptional and epigenetic characteristics akin to meningeal macrophages, likely due to environmental imprinting within the meningeal space. Utilizing parabiosis and skull transplantation, we demonstrated that monocytes from both peripheral blood and skull bone marrow can repopulate microglia-depleted brains. Our results reveal the heterogeneous origins and cellular dynamics of brain parenchymal macrophages at baseline and in models of microglia depletion.

Amplification of autoimmune organ damage by NKp46-activated ILC1s.

In systemic lupus erythematosus, loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here we set out to dissect layers and hierarchies of autoimmune kidney inflammation to identify tissue-specific cellular hubs that amplify autoinflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blockade and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILCs) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signalling in a distinct subset of group 1 ILCs (ILC1s) instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILCs promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody clone mNCR1.15; ref. 2) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data provide support for the idea that NKp46+ ILC1s promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1s therefore constitutes a previously unrecognized, crucial tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.

Western diet reduces small intestinal intraepithelial lymphocytes via FXR-Interferon pathway.

The prevalence of obesity in the United States has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar "Western" diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed gut adaptive immunity. Using small intestinal intraepithelial lymphocytes (IEL) as a readout, we found that in non-inflammatory bowel disease (IBD) subjects, high body mass index correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to reduce IEL but not splenic, blood, or bone marrow lymphocytes, and the effect was reversible after another 2 weeks of standard diet (SD) washout. Importantly, WD-associated IEL reduction was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL reduction. We further found that WD-mediated Farnesoid X Receptor (FXR) activation in the gut triggered IEL reduction, and this was partially mediated by intestinal phagocytes. Activated FXR signaling stimulated phagocytes to secrete type I IFN, and inhibition of either FXR or type I IFN signaling within the phagocytes prevented WD-mediated IEL loss. Therefore, WD consumption represses both innate and adaptive immunity in the gut. These findings have significant clinical implications in the understanding of how diet modulates mucosal immunity.

Neoantigen-specific cytotoxic Tr1 CD4 T cells suppress cancer immunotherapy.

CD4+ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses1-5, other CD4+ T cells have recently been implicated in inhibiting this response6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Mucosal Single-Cell Profiling of Crohn's-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets.

BACKGROUND & AIMS: The pathophysiology of Crohn's-like disease of the pouch (CDP) in patients with a history of ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single-cell analyses. METHODS: Endoscopic samples were collected from pouch body and prepouch ileum (pouch/ileum) of 50 patients with an ileal pouch-anal anastomosis. Single-cell RNA sequencing was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with UC with normal pouch/ileum, CDP, pouchitis, and those with familial adenomatous polyposis after pouch formation. Findings were independently validated using immunohistochemistry. RESULTS: The cell populations/states in the pouch body differed from those in the prepouch ileum, likely secondary to increased microbial burden. Compared with the familial adenomatous polyposis pouch, the UC pouch was enriched in colitogenic immune cells even without inflammation. CDP was characterized by increases in T helper 17 cells, inflammatory fibroblasts, inflammatory monocytes, TREM1+ monocytes, clonal expansion of effector T cells, and overexpression of T helper 17 cells-inducing cytokine genes such as IL23, IL1B, and IL6 by mononuclear phagocytes. Ligand-receptor analysis further revealed a stromal-mononuclear phagocytes-lymphocyte circuit in CDP. Integrated analysis showed that up-regulated immune mediators in CDP were similar to those in CD and pouchitis, but not UC. Additionally, CDP pouch/ileum exhibited heightened endoplasmic reticulum stress across all major cell compartments. CONCLUSIONS: CDP likely represents a distinct entity of inflammatory bowel disease with heightened endoplasmic reticulum stress in both immune and nonimmune cells, which may become a novel diagnostic biomarker and therapeutic target for CDP.

High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Diversity of group 1 innate lymphoid cells in human tissues.

Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES- ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7+PRDM1- ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683+ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases.

TREM2 protects from atherosclerosis by limiting necrotic core formation.

Atherosclerosis is a chronic disease of the vascular wall driven by lipid accumulation and inflammation in the intimal layer of arteries, and its main complications, myocardial infarction and stroke, are the leading cause of mortality worldwide [1], [2]. Recent studies have identified Triggering receptor expressed on myeloid cells 2 (TREM2), a lipid-sensing receptor regulating myeloid cell functions [3], to be highly expressed in macrophage foam cells in experimental and human atherosclerosis [4]. However, the role of TREM2 in atherosclerosis is not fully known. Here, we show that hematopoietic or global TREM2 deficiency increased, whereas TREM2 agonism decreased necrotic core formation in early atherosclerosis. We demonstrate that TREM2 is essential for the efferocytosis capacities of macrophages, and to the survival of lipid-laden macrophages, indicating a crucial role of TREM2 in maintaining the balance between foam cell death and clearance of dead cells in atherosclerotic lesions, thereby controlling plaque necrosis.

The centrosomal protein FGFR1OP controls myosin function in murine intestinal epithelial cells.

Recent advances in human genetics have shed light on the genetic factors contributing to inflammatory diseases, particularly Crohn's disease (CD), a prominent form of inflammatory bowel disease. Certain risk genes associated with CD directly influence cytokine biology and cell-specific communication networks. Current CD therapies primarily rely on anti-inflammatory drugs, which are inconsistently effective and lack strategies for promoting epithelial restoration and mucosal balance. To understand CD's underlying mechanisms, we investigated the link between CD and the FGFR1OP gene, which encodes a centrosome protein. FGFR1OP deletion in mouse intestinal epithelial cells disrupted crypt architecture, resulting in crypt loss, inflammation, and fatality. FGFR1OP insufficiency hindered epithelial resilience during colitis. FGFR1OP was crucial for preserving non-muscle myosin II activity, ensuring the integrity of the actomyosin cytoskeleton and crypt cell adhesion. This role of FGFR1OP suggests that its deficiency in genetically predisposed individuals may reduce epithelial renewal capacity, heightening susceptibility to inflammation and disease.

TREM2 deficiency reprograms intestinal macrophages and microbiota to enhance anti-PD-1 tumor immunotherapy.

The gut microbiota and tumor-associated macrophages (TAMs) affect tumor responses to anti-programmed cell death protein 1 (PD-1) immune checkpoint blockade. Reprogramming TAM by either blocking or deleting the macrophage receptor triggering receptor on myeloid cells 2 (TREM2) attenuates tumor growth, and lack of functional TREM2 enhances tumor elimination by anti-PD-1. Here, we found that anti-PD-1 treatment combined with TREM2 deficiency in mice induces proinflammatory programs in intestinal macrophages and a concomitant expansion of Ruminococcus gnavus in the gut microbiota. Gavage of wild-type mice with R. gnavus enhanced anti-PD-1-mediated tumor elimination, recapitulating the effect occurring in the absence of TREM2. A proinflammatory intestinal environment coincided with expansion, increased circulation, and migration of TNF-producing CD4+ T cells to the tumor bed. Thus, TREM2 remotely controls anti-PD-1 immune checkpoint blockade through modulation of the intestinal immune environment and microbiota, with R. gnavus emerging as a potential probiotic agent for increasing responsiveness to anti-PD-1.

An inducible genetic tool to track and manipulate specific microglial states reveals their plasticity and roles in remyelination.

Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative-region-associated microglia (PAMs) in developing white matter and disease-associated microglia (DAMs) prevalent in various neurodegenerative conditions. PAMs and DAMs share a similar core gene signature. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we generated an inducible Cre driver line, Clec7a-CreERT2, that targets PAMs and DAMs in the brain parenchyma. Utilizing this tool, we profiled labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM and DAM gene expression. Through long-term tracking, we demonstrated microglial state plasticity. Lastly, we specifically depleted DAMs in demyelination, revealing their roles in disease recovery. Together, we provide a versatile genetic tool to characterize microglial states in CNS development and disease.

Progenitors of distinct lineages shape the diversity of mature type 2 conventional dendritic cells.

Conventional dendritic cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2, responsible for priming naive CD8+ and CD4+ T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300ciCre-hCD2R26tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompasses myeloid-derived pre-cDC2 and lymphoid-derived plasmacytoid DC (pDC)-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared with Ms4a3CreR26tdTomato lineage-traced DC3, a monocyte-DC progenitor (MDP)-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to T cell-dependent antigens. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 across tissues. Thus, ontogenesis may impact tissue immune responses.

The IRE1α/XBP1 pathway sustains cytokine responses of group 3 innate lymphoid cells in inflammatory bowel disease.

Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. ER stress is linked to inflammatory bowel disease (IBD). Here, we used cell culture, mouse models, and human specimens to determine whether ER stress in ILC3s affects IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24-hour rhythmic expression pattern of the master ER stress response regulator inositol-requiring kinase 1α/X-box-binding protein 1 (IRE1α/XBP1). Proinflammatory cytokine IL-23 selectively stimulated IRE1α/XBP1 in mouse ILC3s through mitochondrial ROS (mtROS). IRE1α/XBP1 was activated in ILC3s from mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s (Ire1αΔRorc) showed reduced expression of the ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s+ ILC3s in patients with Crohn's disease before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with the response to treatment. We demonstrate that a noncanonical mtROS-IRE1α/XBP1 pathway augmented cytokine production by ILC3s and identify XBP1s+ ILC3s as a potential biomarker for predicting the response to anti-IL-23 therapies in IBD.

Antibody-mediated targeting of human microglial leukocyte Ig-like receptor B4 attenuates amyloid pathology in a mouse model.

Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-ß (Aß) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aß and carry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping around Aß plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aß load, mitigated some Aß-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

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.

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.

Type 2 cannabinoid receptor expression on microglial cells regulates neuroinflammation during graft-versus-host disease.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype that potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and have implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

Reviewing the Clostridioides difficile Mouse Model: Insights into Infection Mechanisms.

Clostridioides difficile is an anaerobic, spore-forming bacterium associated with intestinal infection, manifesting a broad spectrum of gastrointestinal symptoms, ranging from mild diarrhea to severe colitis. A primary risk factor for the development of C. difficile infection (CDI) is antibiotic exposure. Elderly and immunocompromised individuals are particularly vulnerable to CDI. A pivotal aspect for comprehending the complexities of this infection relies on the utilization of experimental models that mimic human CDI transmission, pathogenesis, and progression. These models offer invaluable insights into host-pathogen interactions and disease dynamics, and serve as essential tools for testing potential therapeutic approaches. In this review, we examine the animal model for CDI and delineate the stages of infection, with a specific focus on mice. Our objective is to offer an updated description of experimental models employed in the study of CDI, emphasizing both their strengths and limitations.