Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them.

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

Alzheimer's disease (AD) is the most common form of dementia, and neuroinflammation is an important hallmark of the pathogenesis. Tumor necrosis factor (TNF) might be detrimental in AD, though the results coming from clinical trials on anti-TNF inhibitors are inconclusive. TNFR1, one of the TNF signaling receptors, contributes to the pathogenesis of AD by mediating neuronal cell death. The blood-cerebrospinal fluid (CSF) barrier consists of a monolayer of choroid plexus epithelial (CPE) cells, and AD is associated with changes in CPE cell morphology. Here, we report that TNF is the main inflammatory upstream mediator in choroid plexus tissue in AD patients. This was confirmed in two murine AD models: transgenic APP/PS1 mice and intracerebroventricular (icv) AβO injection. TNFR1 contributes to the morphological damage of CPE cells in AD, and TNFR1 abrogation reduces brain inflammation and prevents blood-CSF barrier impairment. In APP/PS1 transgenic mice, TNFR1 deficiency ameliorated amyloidosis. Ultimately, genetic and pharmacological blockage of TNFR1 rescued from the induced cognitive impairments. Our data indicate that TNFR1 is a promising therapeutic target for AD treatment.

Epidermal keratinocytes undergo a unique form of terminal differentiation and programmed cell death known as cornification. Cornification leads to the formation of the outermost skin barrier, i.e. the cornified layer, as well as to the formation of hair and nails. Different genes are expressed in coordinated waves to provide the structural and regulatory components of cornification. Differentiation-associated keratin intermediate filaments form a complex scaffold accumulating in the cytoplasm and, upon removal of cell organelles, fill the entire cell interior mainly to provide mechanical strength. In addition, a defined set of proteins is cross-linked by transglutamination in the cell periphery to form the so-called cornified envelope. Extracellular modifications include degradation of the tight linkages between corneocytes by excreted proteases, which allows corneocyte shedding by desquamation, and stacking and modification of the excreted lipids that fill the intercellular spaces between corneocytes to provide a water-repellant barrier. In hard skin appendages such as hair and nails these tight intercorneocyte connections remain permanent. Various lines of evidence exist for a role of organelle disintegration, proteases, nucleases, and transglutaminases contributing to the actual cell death event. However, many mechanistic aspects of kearatinocyte death during cornification remain elusive. Importantly, it has recently become clear that keratinocytes activate anti-apoptotic and anti-necroptotic pathways to prevent premature cell death during terminal differentiation. This review gives an overview of the current concept of cornification as a mode of programmed cell death and the anti-cell death mechanisms in the epidermis that secure epidermal homeostasis. This article is part of a Special Section entitled: Cell Death Pathways.

Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

The root cap is a plant organ that ensheathes the meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing constant cell generation by specific stem cells with the disposal of differentiated cells at the root cap edge. This cellular turnover is critical for the maintenance of root cap size and its position around the growing root tip, but how this is achieved and controlled in the model plant Arabidopsis thaliana remains subject to contradictory hypotheses.

In humans, receptor-interacting protein kinase 4 (RIPK4) mutations can lead to the autosomal recessive Bartsocas-Papas and popliteal pterygium syndromes, which are characterized by severe skin defects, pterygia, as well as clefting. We show here that the epithelial fusions observed in RIPK4 full knockout (KO) mice are E-cadherin dependent, as keratinocyte-specific deletion of E-cadherin in RIPK4 full KO mice rescued the tail-to-body fusion and fusion of oral epithelia. To elucidate RIPK4 function in epidermal differentiation and development, we generated epidermis-specific RIPK4 KO mice (RIPK4EKO). In contrast to RIPK4 full KO epidermis, RIPK4EKO epidermis was normally stratified and the outside-in skin barrier in RIPK4EKO mice was largely intact at the trunk, in contrast to the skin covering the head and the outer end of the extremities. However, RIPK4EKO mice die shortly after birth due to excessive water loss because of loss of tight junction protein claudin-1 localization at the cell membrane, which results in tight junction leakiness. In contrast, mice with keratinocyte-specific RIPK4 deletion during adult life remain viable. Furthermore, our data indicate that epidermis-specific deletion of RIPK4 results in delayed keratinization and stratum corneum maturation and altered lipid organization and is thus indispensable during embryonic development for the formation of a functional inside-out epidermal barrier.

Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting memory and cognition. The disease is accompanied by an abnormal deposition of ß-amyloid plaques in the brain that contributes to neurodegeneration and is known to induce glial inflammation. Studies in the APP/PS1 mouse model of ß-amyloid-induced neuropathology have suggested a role for inflammasome activation in ß-amyloid-induced neuroinflammation and neuropathology.

The RIP kinases have emerged as essential mediators of cellular stress that integrate both extracellular stimuli emanating from various cell-surface receptors and signals coming from intracellular pattern recognition receptors. The molecular mechanisms regulating the ability of the RIP proteins to transduce the stress signals remain poorly understood, but seem to rely only partially on their kinase activities. Recent studies on RIP1 and RIP2 have highlighted the importance of ubiquitination as a key process regulating their capacity to activate downstream signaling pathways. In this study, we found that XIAP, cIAP1 and cIAP2 not only directly bind to RIP1 and RIP2 but also to RIP3 and RIP4. We show that cIAP1 and cIAP2 are direct E3 ubiquitin ligases for all four RIP proteins and that cIAP1 is capable of conjugating the RIPs with diverse types of ubiquitin chains, including linear chains. Consistently, we show that repressing cIAP1/2 levels affects the activation of NF-κB that is dependent on RIP1, -2, -3 and -4. Finally, we identified Lys51 and Lys145 of RIP4 as two critical residues for cIAP1-mediated ubiquitination and NF-κB activation.

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-κB) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microglia-confined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS)-like disease, due to hyperactivation of the Nlrp3 inflammasome leading to enhanced interleukin-1β secretion and CNS inflammation. Finally, we confirm a Nlrp3 inflammasome signature and IL-1β expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.

There is a growing appreciation that membrane-bound organelles in eukaryotic cells communicate directly with one another through direct membrane contact sites. Mitochondria-associated membranes are specialized subdomains of the endoplasmic reticulum that function as membrane contact sites between the endoplasmic reticulum and mitochondria. These sites have emerged as major players in lipid metabolism and calcium signaling. More recently also autophagy and mitochondrial dynamics have been found to be regulated at ER-mitochondria contact sites. Neurons critically depend on mitochondria-associated membranes as a means to exchange metabolites and signaling molecules between these organelles. This is underscored by the fact that genes affecting mitochondrial and endoplasmic reticulum homeostasis are clearly overrepresented in several hereditary neurodegenerative disorders. Conversely, the processes affected by the contact sites between the endoplasmic reticulum and mitochondria are widely implicated in neurodegeneration. This review will focus on the most recent data addressing the structural composition and function of the mitochondria-associated membranes. In addition, the 3D morphology of the contact sites as observed using volume electron microscopy is discussed. Finally, it will highlight the role of several key proteins associated with these contact sites that are involved not only in dementias, amyotrophic lateral sclerosis and Parkinson's disease, but also in axonopathies such as hereditary spastic paraplegia and Charcot-Marie-Tooth disease.

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins.IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

The recent advent of 3D in electron microscopy (EM) has allowed for detection of nanometer resolution structures. This has caused an explosion in dataset size, necessitating the development of automated workflows. Moreover, large 3D EM datasets typically require hours to days to be acquired and accelerated imaging typically results in noisy data. Advanced denoising techniques can alleviate this, but tend to be less accessible to the community due to low-level programming environments, complex parameter tuning or a computational bottleneck. We present DenoisEM: an interactive and GPU accelerated denoising plugin for ImageJ that ensures fast parameter tuning and processing through parallel computing. Experimental results show that DenoisEM is one order of magnitude faster than related software and can accelerate data acquisition by a factor of 4 without significantly affecting data quality. Lastly, we show that image denoising benefits visualization and (semi-)automated segmentation and analysis of ultrastructure in various volume EM datasets.

In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by the accumulation of amyloid β (Aβ) and neurofibrillary tangles. The last decade, it became increasingly clear that neuroinflammation plays a key role in both the initiation and progression of AD. Moreover, also the presence of peripheral inflammation has been extensively documented. However, it is still ambiguous whether this observed inflammation is cause or consequence of AD pathogenesis. Recently, this has been studied using amyloid precursor protein (APP) overexpression mouse models of AD. However, the findings might be confounded by APP-overexpression artifacts. Here, we investigated the effect of low-grade peripheral inflammation in the APP knock-in (AppNL-G-F) mouse model. This revealed that low-grade peripheral inflammation affects (1) microglia characteristics, (2) blood-cerebrospinal fluid barrier integrity, (3) peripheral immune cell infiltration and (4) Aβ deposition in the brain. Next, we identified mechanisms that might cause this effect on AD pathology, more precisely Aβ efflux, persistent microglial activation and insufficient Aβ clearance, neuronal dysfunction and promotion of Aβ aggregation. Our results further strengthen the believe that even low-grade peripheral inflammation has detrimental effects on AD progression and may further reinforce the idea to modulate peripheral inflammation as a therapeutic strategy for AD.