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Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogen Cryptococcus neoformans in 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Marco -/- macrophages also showed elevated vomocytosis of a yeast-locked C. albicans strain, suggesting this to be a broadly relevant observation. We go on to show that MARCO's role in modulating vomocytosis is independent of its role as a phagocytic receptor, suggesting that this protein may play an important and hitherto unrecognised role in modulating macrophage behaviour., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

Escherichia coli ( E. coli ) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7-a commonly used experimental murine macrophage model-to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αβ) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients., Methods: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-β was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αβ, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αβR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction., Results: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αβ by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αβ-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αβ-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-β pretreatment enhances the subsequent induction of IFN-αβ in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αβ overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice., Conclusion: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4 -/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4 -/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans., (© 2023. Springer Nature Limited.)

Space and time can each act as scaffolds for the individuation and selection of visual objects in working memory. Here we ask whether there is a trade-off between the use of space and time for visual working memory: whether observers will rely less on space, when memoranda can additionally be individuated through time. We tracked the use of space through directional biases in microsaccades after attention was directed to memory contents that had been encoded simultaneously or sequentially to the left and right of fixation. We found that spatial gaze biases were preserved when participants could (Experiment 1) and even when they had to (Experiment 2) additionally rely on time for object individuation. Thus, space remains a profound organizing medium for working memory even when other organizing sources are available and utilized, with no evidence for an obligatory trade-off between the use of space and time., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2023.)

Micro and nanosize plastic polymers degrading from large plastic compounds are accumulating in the natural environment and expose potential biological threats to human health. These particles are largely persistent and consequently accumulate in the exposed individuals. The presence of microplastics has already been demonstrated in various human organs including the lung, the gastrointestinal system and the blood raising concerns about their possible harmful effects. The chemical composition, size and shape of microplastics as well as their weathering status represent important factors influencing the potential impact of microplastics on tissues. In addition, microplastics can function as vectors for adsorbed chemical compounds and may harbour and deliver live microbial pathogens or their ligands. In vitro and in vivo animal studies demonstrated that microplastics are taken up to cells in a size and cell type dependent manner. Once inside the targeted cell they activate oxidative processes, mitochondrial dysfunction and ER-stress. These molecular processes result in the activation or repression of cell type specific functions and potentially in the induction of cytotoxicity. The microplastic elicited events may result in inflammation, organ damage and fibrosis of the targeted organs as well as in systemic immunological and metabolic conditions. In addition, microplastics may impact on the gut microbiota which may exert further gastrointestinal and systemic metabolic and immunological effects. In this minireview, we evaluate the factors and mechanisms that influence potential microplastic induced cellular and organ pathologies in humans and discuss limitations of current understanding regarding microplastic elicited conditions as well as future perspectives for research., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Inflammation is central to several diseases. TLR4 mediates inflammation by recognising and binding to bacterial lipopolysaccharides and interacting with other proteins in the TLR4 signalling pathway. Although there is extensive research on TLR4-mediated inflammation, there are gaps in understanding its mechanisms. Recently, TLR4 co-localised with LPCAT2, a lysophospholipid acetyltransferase. LPCAT2 is already known to influence lipopolysaccharide-induced inflammation; however, the mechanism of LPCAT2 influencing lipopolysaccharide-mediated inflammation is not understood. The present study combined computational analysis with biochemical analysis to investigate the influence of LPCAT2 on lysine acetylation in LPS-treated RAW264.7 cells. The results suggest for the first time that LPCAT2 influences lysine acetylation in LPS-treated RAW264.7 cells. Moreover, we detected acetylated lysine residues on TLR4. The present study lays a foundation for further research on the role of lysine acetylation on TLR4 signalling. Moreover, further research is required to characterise LPCAT2 as a protein acetyltransferase., (© 2022 The Author(s).)

There is an increased societal trend to engage in microdosing, in which small sub-hallucinogenic amounts of psychedelics are consumed on a regular basis. Following subjective reports that microdosing enhances the experience of nature and art, in the present study we set out to study the effects of psilocybin microdosing on feelings of awe and art perception. In this preregistered combined field- and lab-based study, participants took part in a microdosing workshop after which they volunteered to self-administer a psilocybin microdose or a placebo for three consecutive weeks, while the condition was kept blind to the participants and researchers. Following a 2-week break, the condition assignment was reversed. During each block, participants visited the lab twice to measure the effects of psilocybin microdosing vs. placebo. We used standardized measures of awe, in which participants reported their experiences in response to short videos or when viewing abstract artworks from different painters. Our confirmatory analyses showed that participants felt more awe in response to videos representing funny animals and moving objects in the microdosing compared to the placebo condition. However, about two-third of our participants were breaking blind to their experimental condition. Our exploratory findings suggest that expectancy-effects may be a driving factor underlying the subjective benefits of microdosing., (© 2021. The Author(s).)

Background: Microdoses of psychedelics (i.e. a sub-hallucinogenic dose taken every third day) can reduce symptoms of depression, anxiety and stress according to anecdotal reports and observational studies. Research with medium to high doses of psilocybin points towards potential underlying mechanisms, including the modulation of emotion and interoceptive processing., Aims: In this preregistered study, we investigated whether psilocybin microdoses alter self-reported interoceptive awareness and whether repeated microdosing over 3 weeks modulates emotion processing and reduces symptoms of anxiety and depression., Methods: We used a double-blind, placebo-controlled, within-subject crossover design. Participants completed the Multidimensional Assessment of Interoceptive Awareness Questionnaire 1½ h after self-administering their second dose (or placebo), and the emotional go/no-go task and the shortened Depression Anxiety Stress Scale 1½ h after self-administering their seventh dose., Results: Our confirmatory analyses revealed that psilocybin microdosing did not affect emotion processing or symptoms of anxiety and depression compared with placebo. Our exploratory analyses revealed that psilocybin microdosing did not affect self-reported interoceptive awareness, that symptoms of depression and stress were significantly reduced in the first block compared with baseline, that participants broke blind in the second block and that there was no effect of expectations. Further research in a substance-naïve population with clinical range anxiety and depressive symptoms is needed to substantiate the potential beneficial effects of microdosing.

Oropouche virus (OROV) is responsible for outbreaks of Oropouche fever in parts of South America. We recently identified and isolated OROV from a febrile Ecuadorian patient, however, a previously published qRT-PCR assay did not detect OROV in the patient sample. A primer mismatch to the Ecuadorian OROV lineage was identified from metagenomic sequencing data. We report the optimisation of an qRT-PCR assay for the Ecuadorian OROV lineage, which subsequently identified a further five cases in a cohort of 196 febrile patients. We isolated OROV via cell culture and developed an algorithmically-designed primer set for whole-genome amplification of the virus. Metagenomic sequencing of the patient samples provided OROV genome coverage ranging from 68-99%. The additional cases formed a single phylogenetic cluster together with the initial case. OROV should be considered as a differential diagnosis for Ecuadorian patients with febrile illness to avoid mis-diagnosis with other circulating pathogens., Competing Interests: The authors have declared that no competing interests exist.

Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.

Contaminated marine bathing water has been reported to adversely affect human health. Our data demonstrated a correlation between total endotoxin (lipopolysaccharide; LPS) levels and degree of contamination of marine bathing waters. To assess the potential health implications of LPS present in marine bathing waters, the inflammation-inducing potency of water samples collected at different time points at multiple sampling sites were assessed using a cell culture-based assay. The numbers of fecal indicator bacteria (FIB) were also examined in the same samples. Water samples were used to stimulate two cell culture models: (1) a novel non-transformed continuously growing murine cell line Max Plank Institute (MPI) characteristic of alveolar macrophages and (2) human MonoMac 6 monocyte cell line. The inflammatory potential of the samples was assessed by measuring the release of inflammatory cytokines. The presence of high levels of LPS in contaminated bathing water led to induction of inflammatory response from our in vitro cell-based bioassays suggesting its potential health impact. This finding introduces an in vitro culture assay that reflects the level of LPS in water samples. These observations further promote previous finding that LPS is a reliable surrogate biomarker for fecal contamination of bathing water.

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis ( Mtb ) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host- Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1β, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb . By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

The enzyme tartrate resistant acid phosphatase (TRAP, two isoforms 5a and 5b) is highly expressed in alveolar macrophages, but its function there is unclear and potent selective inhibitors of TRAP are required to assess functional aspects of the protein. We found higher TRAP activity/expression in lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma compared to controls and more TRAP activity in lungs of mice with experimental COPD or asthma. Stimuli related to asthma and/or COPD were tested for their capacity to induce TRAP. Receptor activator of NF-κb ligand (RANKL) and Xanthine/Xanthine Oxidase induced TRAP mRNA expression in mouse macrophages, but only RANKL also induced TRAP activity in mouse lung slices. Several Au(III) coordination compounds were tested for their ability to inhibit TRAP activity and [Au(4,4'-dimethoxy-2,2'-bipyridine)Cl 2 ][PF 6 ] (AubipyOMe) was found to be the most potent inhibitor of TRAP5a and 5b activity reported to date (IC50 1.3 and 1.8 μM respectively). AubipyOMe also inhibited TRAP activity in murine macrophage and human lung tissue extracts. In a functional assay with physiological TRAP substrate osteopontin, AubipyOMe inhibited mouse macrophage migration over osteopontin-coated membranes. In conclusion, higher TRAP expression/activity are associated with COPD and asthma and TRAP is involved in regulating macrophage migration.

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors., (Copyright © 2017 Maler et al.)

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular "pulse" of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection.

Mononuclear phagocytes have been viewed for a long time as one distinct lineage where continuous division of haematopoietic progenitor cells give rise to and replenish differentiated mature cells with a limited life-span. Very recent data have demonstrated however, that in addition to this, proliferation of differentiated macrophages of mostly embryonic origin also contribute significantly to the mononuclear phagocyte system. Recently developed primary tissue culture models of self-renewing differentiated resident macrophages are now available to facilitate our understanding of macrophage heterogeneity and to provide special tools to study general and specific macrophage functions as well. In this review, we will focus on current knowledge on the concept of self-renewing macrophages and discuss aspects of their origin, development and function., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Adenoviruses are important infectious agents and also emerging vectors in different biomedical applications. These viruses elicit a strong innate and adaptive immune response, which influences both the course of disease and the success of the applied vectors. Several Toll-like Receptor (TLR)-dependent and -independent mechanisms contribute to these responses. Understanding of the involved viral and cellular factors is crucial for the treatment of various adenovirus diseases and the optimal design of adenovirus vector applications. Here we summarize our current understanding of the complex nature of adenovirus-induced innate immune mechanisms.

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α(+) conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α(+) cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α(+) cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α(+) cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α(+) DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α(+) cDCs and their human equivalents.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus. It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-alpha/beta) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-alpha) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-alpha/beta by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-alpha/beta release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-alpha release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-alpha by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-beta production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-alpha/beta production.

Allergic contact hypersensitivity (CHS) is a T cell-mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rbeta2-deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12-independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) beta2, or both, we show that the concomitant absence of TLR4 and IL-12Rbeta2, but not the absence of TLR4 or IL-12Rbeta2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rbeta2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12-independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12-competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rbeta2-deficient mice, but not in germ-free TLR4/IL-12Rbeta2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Transcripts for the Epstein-Barr virus (EBV)-encoded nuclear antigens are initiated at the alternative promoters Wp, Cp and Qp. Although the host cell-dependent activity of Cp is regulated by DNA methylation, Qp is unmethylated independently of its activity. Because histone modifications affect the chromatin structure, we compared the levels of diacetylated histone H3, tetraacetylated histone H4 and histone H3 dimethylated on lysine 4 (H3K4me2) at Cp and Qp, in well characterized cell lines representing the major EBV latency types. We found an activity-dependent histone code: acetylated histones marked active Cp, whereas active Qp was selectively enriched both in acetylated histones and H3K4me2. We concluded that active (but not silent) Cp and Qp are located to 'acetylation islands' in latent, episomal EBV genomes, similar to the active chromatin domains of the human genome.

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-alpha and IFN-gamma in response to LPS, lipid A, synthetic lipopeptide Pam(3)CysK(4), or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9(-/-), mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9(-/-) mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-gamma is a key mediator of these effects, and enhanced IFN-gamma mRNA expression was absent in spleen and liver of P. acnes-primed TLR9(-/-) mice, we conclude that TLR9 is required for the induction of IFN-gamma by P. acnes.

The cell surface HLA-DR molecule binds foreign peptide antigen and forms an intercellular complex with the T cell receptor in the course of the development of an immune response against or immune tolerance to the antigen represented by the bound peptide. The HLA-DR molecule also functions as a receptor that mediates cell signaling pathways, including as yet poorly characterized pathway(s) leading to apoptosis. Expression of HLA-DR mRNA and protein is ordinarily inducible by interferon-gamma but is not inducible in tumor cells defective for the retinoblastoma tumor suppressor protein (Rb). In the case of the HLA-DRA gene, which encodes the HLA-DR heavy chain, previous work has indicated that this loss of inducibility is attributable to Oct-1 binding to the HLA-DRA promoter. In this report, we used Oct-1 antisense transformants to determine that Oct-1 represses the interferon-gamma response of the endogenous HLA-DRA gene. This determination is consistent with results from a chromatin immunoprecipitation assay, indicating that Oct-1 occupies the endogenous HLA-DRA promoter when the HLA-DRA promoter is inactive in Rb-defective cells but not when the promoter is converted to a previously defined, transcriptionally competent state, induced by treatment of the Rb-defective cells with the HDAC inhibitor, trichostatin A. In vitro DNA-protein binding analyses indicated that Oct-1 prevents HLA-DRA promoter activation by mediating the formation of a complex of proteins, termed DRAN (DRA negative), that blocks NF-Y access to the promoter.

Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.

Post-translational modifications of histones, in general, and acetylation/deacetylation, in particular, can dramatically alter gene expression in eukaryotic cells. In humans, four highly homologous class I HDAC enzymes (HDAC1, HDAC2, HDAC3, and HDAC8) have been identified to date. Although HDAC3 shares some structural and functional similarities with other class I HDACs, it exists in multisubunit complexes separate and different from other known HDAC complexes, implying that individual HDACs might function in a distinct manner. In this current study, to understand further the cellular function of HDAC3 and to uncover possible unique roles this protein may have in gene regulation, we performed a detailed analysis of HDAC3 using deletion mutations. Surprisingly, we found that the non-conserved C-terminal region of HDAC3 is required for both deacetylase and transcriptional repression activity. In addition, we discovered that the central portion of the HDAC3 protein possesses a nuclear export signal, whereas the C-terminal part of HDAC3 contributes to the protein's localization in the nucleus. Finally, we found that HDAC3 forms oligomers in vitro and in vivo and that the N-terminal portion of HDAC3 is necessary for this property. These data indicate that HDAC3 comprises separate, non-overlapping domains that contribute to the unique properties and function of this protein.