Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase.

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.

Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport.

Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.

High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3.

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

NLRP3 inflammasome activation drives tau pathology.

Alzheimer's disease is characterized by the accumulation of amyloid-beta in plaques, aggregation of hyperphosphorylated tau in neurofibrillary tangles and neuroinflammation, together resulting in neurodegeneration and cognitive decline1. The NLRP3 inflammasome assembles inside of microglia on activation, leading to increased cleavage and activity of caspase-1 and downstream interleukin-1ß release2. Although the NLRP3 inflammasome has been shown to be essential for the development and progression of amyloid-beta pathology in mice3, the precise effect on tau pathology remains unknown. Here we show that loss of NLRP3 inflammasome function reduced tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. Tau activated the NLRP3 inflammasome and intracerebral injection of fibrillar amyloid-beta-containing brain homogenates induced tau pathology in an NLRP3-dependent manner. These data identify an important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer's disease, demonstrating that neurofibrillary tangles develop downstream of amyloid-beta-induced microglial activation.

Differential impact of high-salt levels in vitro and in vivo on macrophage core functions.

The consumption of processed food is on the rise leading to huge intake of excess dietary salt, which strongly correlates with development of hypertension, often leading to cardiovascular diseases such as stroke and heart attack, as well as activation of the immune system. The effect of salt on macrophages is especially interesting as they are able to sense high sodium levels in tissues leading to transcriptional changes. In the skin, macrophages were shown to influence lymphatic vessel growth which, in turn, enables the transport of excess salt and thereby prevents the development of high blood pressure. Furthermore, salt storage in the skin has been linked to the onset of pro-inflammatory effector functions of macrophages in pathogen defence. However, there is only little known about the mechanisms which are involved in changing macrophage function to salt exposure. Here, we characterize the response of macrophages to excess salt both in vitro and in vivo. Our results validate and strengthen the notion that macrophages exhibit chemotactic migration in response to salt gradients in vitro. Furthermore, we demonstrate a reduction in phagocytosis and efferocytosis following acute salt challenge in vitro. While acute exposure to a high-salt diet in vivo has a less pronounced impact on macrophage core functions such as phagocytosis, our data indicate that prolonged salt challenge may exert a distinct effect on the function of macrophages. These findings suggest a potential role for excessive salt sensing by macrophages in the manifestation of diseases related to high-salt diets and explicitly highlight the need for in vivo work to decipher the physiologically relevant impact of excess salt on tissue and cell function.

Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.

Toll-like receptor-3 contributes to the development of aortic valve stenosis.

Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3-/- mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3-/- mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.

Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.

Macrophage activation is associated with profound transcriptional reprogramming. Although much progress has been made in the understanding of macrophage activation, polarization, and function, the transcriptional programs regulating these processes remain poorly characterized. We stimulated human macrophages with diverse activation signals, acquiring a data set of 299 macrophage transcriptomes. Analysis of this data set revealed a spectrum of macrophage activation states extending the current M1 versus M2-polarization model. Network analyses identified central transcriptional regulators associated with all macrophage activation complemented by regulators related to stimulus-specific programs. Applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients. Finally, by integrating murine data from the ImmGen project we propose a refined, activation-independent core signature for human and murine macrophages. This resource serves as a framework for future research into regulation of macrophage activation in health and disease.

Natural Killer Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Against SARS-CoV-2 After Natural Infection Is More Potent Than After Vaccination.

We compared the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific antibodies to induce natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC) in patients with natural infection and vaccinated persons. Analyzing plasma samples from 39 coronavirus disease 2019 (COVID-19) patients and 11 vaccinated individuals, significant induction of ADCC could be observed over a period of more than 3 months in both vaccinated and recovered individuals. Although plasma antibody concentrations were lower in recovered patients, we found antibodies elicited by natural infection induced a significantly stronger ADCC response compared to those induced by vaccination, which may affect protection conferred by vaccination.

Molecular features of macrophage activation.

Cellular activation is mainly defined as the response of a cell to exogenous signals eventually leading to changes in protein expression and cellular function. Originally, macrophage activation was mainly associated with phagocytic function. Later other effector functions such as cytokine secretion, upregulation of cell surface receptors came into focus. For a while macrophage activation was classified as being either pro-inflammatory or anti-inflammatory and certain signal transduction pathways were associated with these two conditions. Most recent findings on transcriptional and epigenetic level, however, suggest that the molecular features of macrophage activation are significantly more complex. Here, we will introduce a novel and integrative model of macrophage activation. Albeit recognizing that macrophage activation cannot be reduced to nuclear processes, we will focus in this review on the most recent findings concerning transcriptional and epigenetic regulation of macrophage activation. Understanding the complexity of the central regulatory mechanisms in the nucleus will form a foundation for deciphering all the different effector functions that are associated with macrophage activation.

ATF3 Is a Key Regulator of Macrophage IFN Responses.

Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-ß expression, as well as the magnitude of IFN-ß cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-ß via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Additionally, we observed that ATF3 itself is a type I IFN-inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFNß, as well as the expression of genes downstream of IFN signaling.

Optimization of transcription factor binding map accuracy utilizing knockout-mouse models.

Genome-wide assessment of protein-DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify 'hyper ChIPable regions' as another source of false-positive signals. As the use of the KOIN algorithm reduces false-positive results and thereby prevents misinterpretation of ChIP-seq data, it should be considered as the gold standard for future ChIP-seq analyses, particularly when developing ChIP-assays with novel antibody reagents.

Butyrate and propionate are microbial danger signals that activate the NLRP3 inflammasome in human macrophages upon TLR stimulation.

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1ß release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.

Microglia rescue neurons from aggregate-induced neuronal dysfunction and death through tunneling nanotubes.

Microglia are crucial for maintaining brain health and neuron function. Here, we report that microglia establish connections with neurons using tunneling nanotubes (TNTs) in both physiological and pathological conditions. These TNTs facilitate the rapid exchange of organelles, vesicles, and proteins. In neurodegenerative diseases like Parkinson's and Alzheimer's disease, toxic aggregates of alpha-synuclein (α-syn) and tau accumulate within neurons. Our research demonstrates that microglia use TNTs to extract neurons from these aggregates, restoring neuronal health. Additionally, microglia share their healthy mitochondria with burdened neurons, reducing oxidative stress and normalizing gene expression. Disrupting mitochondrial function with antimycin A before TNT formation eliminates this neuroprotection. Moreover, co-culturing neurons with microglia and promoting TNT formation rescues suppressed neuronal activity caused by α-syn or tau aggregates. Notably, TNT-mediated aggregate transfer is compromised in microglia carrying Lrrk22(Gly2019Ser) or Trem2(T66M) and (R47H) mutations, suggesting a role in the pathology of these gene variants in neurodegenerative diseases.

UNC93B1 variants underlie TLR7-dependent autoimmunity.

UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.