Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis.

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.

Differential Modulation of Dendritic Cell Biology by Endogenous and Exogenous Aryl Hydrocarbon Receptor Ligands.

The aryl hydrocarbon receptor (AhR) is a decisive regulatory ligand-dependent transcription factor. It binds highly diverse ligands, which can be categorized as either endogenous or exogenous. Ligand binding activates AhR, which can adjust inflammatory responses by modulating immune cells such as dendritic cells (DCs). However, how different AhR ligand classes impact the phenotype and function of human monocyte-derived DCs (hMoDCs) has not been extensively studied in a comparative manner. We, therefore, tested the effect of the representative compounds Benzo(a)pyrene (BP), 6-formylindolo[3,2-b]carbazole (FICZ), and Indoxyl 3-sulfate (I3S) on DC biology. Thereby, we reveal that BP significantly induces a tolerogenic response in lipopolysaccharide-matured DCs, which is not apparent to the same extent when using FICZ or I3S. While all three ligand classes activate AhR-dependent pathways, BP especially induces the expression of negative immune regulators, and subsequently strongly subverts the T cell stimulatory capacity of DCs. Using the CRISPR/Cas9 strategy we also prove that the regulatory effect of BP is strictly AhR-dependent. These findings imply that AhR ligands contribute differently to DC responses and incite further studies to uncover the mechanisms and molecules which are involved in the induction of different phenotypes and functions in DCs upon AhR activation.

CD83 expressed by macrophages is an important immune checkpoint molecule for the resolution of inflammation.

Excessive macrophage (Mφ) activation results in chronic inflammatory responses or autoimmune diseases. Therefore, identification of novel immune checkpoints on Mφ, which contribute to resolution of inflammation, is crucial for the development of new therapeutic agents. Herein, we identify CD83 as a marker for IL-4 stimulated pro-resolving alternatively activated Mφ (AAM). Using a conditional KO mouse (cKO), we show that CD83 is important for the phenotype and function of pro-resolving Mφ. CD83-deletion in IL-4 stimulated Mφ results in decreased levels of inhibitory receptors, such as CD200R and MSR-1, which correlates with a reduced phagocytic capacity. In addition, CD83-deficient Mφ upon IL-4 stimulation, show an altered STAT-6 phosphorylation pattern, which is characterized by reduced pSTAT-6 levels and expression of the target gene Gata3. Concomitantly, functional studies in IL-4 stimulated CD83 KO Mφ reveal an increased production of pro-inflammatory mediators, such as TNF-α, IL-6, CXCL1 and G-CSF. Furthermore, we show that CD83-deficient Mφ have enhanced capacities to stimulate the proliferation of allo-reactive T cells, which was accompanied by reduced frequencies of Tregs. In addition, we show that CD83 expressed by Mφ is important to limit the inflammatory phase using a full-thickness excision wound healing model, since inflammatory transcripts (e.g. Cxcl1, Il6) were increased, whilst resolving transcripts (e.g. Ym1, Cd200r, Msr-1) were decreased in wounds at day 3 after wound infliction, which reflects the CD83 resolving function on Mφ also in vivo. Consequently, this enhanced inflammatory milieu led to an altered tissue reconstitution after wound infliction. Thus, our data provide evidence that CD83 acts as a gatekeeper for the phenotype and function of pro-resolving Mφ.

CD83 acts as immediate early response gene in activated macrophages and exhibits specific intracellular trafficking properties.

Macrophages (MΦ) are remarkably plastic cells, which assume phenotypes in every shade between a pro-inflammatory classical activation, and anti-inflammatory or resolving activation. Therefore, elucidation of mechanisms involved in shaping MΦ plasticity and function is key to understand their role during immunological balance. The immune-modulating CD83 molecule is expressed on activated immune cells and various tissue resident MΦ, rendering it an interesting candidate for affecting MΦ biology. However, in-depth analyses of the precise kinetics and trafficking of CD83 within pro-inflammatory, LPS activated bone-marrow-derived MΦ have not been performed. In this study, we show that activation with LPS leads to a very fast and strong, but transient increase of CD83 expression on these cells. Its expression peaks within 2 h of stimulation and is thereby faster than the early activation antigen CD69. To trace the CD83 trafficking through MΦs, we employed multiple inhibitors, thereby revealing a de novo synthesis and transport of the protein to the cell surface followed by lysosomal degradation, all within 6 h. Moreover, we found a similar expression kinetic and trafficking in human monocyte derived MΦ. This places CD83 at a very early point of MΦ activation suggesting an important role in decisions regarding the subsequent cellular fate.

HSV-1 Modulates IL-6 Receptor Expression on Human Dendritic Cells.

Dendritic cells (DCs) are the guardians of the immune system since they are located in the majority of peripheral tissues. In addition, they are crucial for the induction of an effective immune response based on their unique capacity to stimulate naive T cells. During co-evolution, the human pathogen herpes simplex virus type 1 (HSV-1) has evolved several immune evasion mechanisms in order to subvert the host's immune system especially by targeting DC biology and function. Here we demonstrate that HSV-1 infection influences the IL-6 receptor (IL6R) expression both on protein and mRNA levels in/on human monocyte-derived mature DCs (mDCs). Surprisingly, reduced IL6R expression levels were also observed on uninfected bystander mDCs. Mechanistically, we clearly show that HSV-1-derived non-infectious light (L-) particles are sufficient to trigger IL6R regulation on uninfected bystander mDCs. These L-particles lack the viral DNA-loaded capsid and are predominantly produced during infection of mDCs. Our results show that the deletion of the HSV-1 tegument protein vhs partially rescued the reduced IL6R surface expression levels on/in bystander mDCs. Using a neutralizing antibody, which perturbs the transfer of L-particles to bystander mDCs, was sufficient to rescue the modulation of IL6R surface expression on uninfected bystander mDCs. This study provides evidence that L-particles transfer specific viral proteins to uninfected bystander mDCs, thereby negatively interfering with their IL6R expression levels, however, to a lesser extend compared to H-particles. Due to their immune-modulatory capacity, L-particles represent an elaborated approach of HSV-1-mediated immune evasion.

Herpes Simplex Virus Type-2 Paralyzes the Function of Monocyte-Derived Dendritic Cells.

Herpes simplex viruses not only infect a variety of different cell types, including dendritic cells (DCs), but also modulate important cellular functions in benefit of the virus. Given the relevance of directed immune cell migration during the initiation of potent antiviral immune responses, interference with DC migration constitutes a sophisticated strategy to hamper antiviral immunity. Notably, recent reports revealed that HSV-1 significantly inhibits DC migration in vitro. Thus, we aimed to investigate whether HSV-2 also modulates distinct hallmarks of DC biology. Here, we demonstrate that HSV-2 negatively interferes with chemokine-dependent in vitro migration capacity of mature DCs (mDCs). Interestingly, rather than mediating the reduction of the cognate chemokine receptor expression early during infection, HSV-2 rapidly induces ß2 integrin (LFA-1)-mediated mDC adhesion and thereby blocks mDC migration. Mechanistically, HSV-2 triggers the proteasomal degradation of the negative regulator of ß2 integrin activity, CYTIP, which causes the constitutive activation of LFA-1 and thus mDC adhesion. In conclusion, our data extend and strengthen recent findings reporting the reduction of mDC migration in the context of a herpesviral infection. We thus hypothesize that hampering antigen delivery to secondary lymphoid organs by inhibition of mDC migration is an evolutionary conserved strategy among distinct members of Herpesviridae.

CD83 orchestrates immunity toward self and non-self in dendritic cells.

Dendritic cells (DCs) are crucial to balance protective immunity and autoimmune inflammatory processes. Expression of CD83 is a well-established marker for mature DCs, although its physiological role is still not completely understood. Using a DC-specific CD83-conditional KO (CD83ΔDC) mouse, we provide new insights into the function of CD83 within this cell type. Interestingly, CD83-deficient DCs produced drastically increased IL-2 levels and displayed higher expression of the costimulatory molecules CD25 and OX40L, which causes superior induction of antigen-specific T cell responses and compromises Treg suppressive functions. This also directly translates into accelerated immune responses in vivo. Upon Salmonella typhimurium and Listeria monocytogenes infection, CD83ΔDC mice cleared both pathogens more efficiently, and CD83-deficient DCs expressed increased IL-12 levels after bacterial encounter. Using the experimental autoimmune encephalomyelitis model, autoimmune inflammation was dramatically aggravated in CD83ΔDC mice while resolution of inflammation was strongly reduced. This phenotype was associated with increased cell influx into the CNS accompanied by elevated Th17 cell numbers. Concomitantly, CD83ΔDC mice had reduced Treg numbers in peripheral lymphoid organs. In summary, we show that CD83 ablation on DCs results in enhanced immune responses by dysregulating tolerance mechanisms and thereby impairing resolution of inflammation, which also demonstrates high clinical relevance.

Endogenous Expression of the Human CD83 Attenuates EAE Symptoms in Humanized Transgenic Mice and Increases the Activity of Regulatory T Cells.

The CD83 is a type I membrane protein and part of the immunoglobulin superfamily of receptors. CD83 is involved in the regulation of antigen presentation and dendritic cell dependent allogeneic T cell proliferation. A soluble form of CD83 inhibits dendritic cell maturation and function. Furthermore, CD83 is expressed on activated B cells, T cells, and in particular on regulatory T cells. Previous studies on murine CD83 demonstrated this molecule to be involved in several immune-regulatory processes, comprising that CD83 plays a key role in the development und function of different immune cells. In order to get further insights into the function of the human CD83 and to provide preclinical tools to guide the function of CD83/sCD83 for therapeutic purposes we generated Bacterial Artificial Chromosomes (BAC) transgenic mice. BACs are excellent tools for manipulating large DNA fragments and are utilized to engineer transgenic mice by pronuclear injection. Two different founders of BAC transgenic mice expressing human CD83 (BAC-hCD83tg mice) were generated and were examined for the hCD83 expression on different immune cells as well as both the in vitro and in vivo role of human CD83 (hCD83) in health and disease. Here, we found the hCD83 molecule to be present on activated DCs, B cells and subtypes of CD4+ T cells. CD8+ T cells, on the other hand, showed almost no hCD83 expression. To address the function of hCD83, we performed in vitro mixed lymphocyte reactions (MLR) as well as suppression assays and we used the in vivo model of experimental autoimmune encephalomyelitis (EAE) comparing wild-type and hCD83-BAC mice. Results herein showed a clearly diminished capacity of hCD83-BAC-derived T cells to proliferate accompanied by an enhanced activation and suppressive activity of hCD83-BAC-derived Tregs. Furthermore, hCD83-BAC mice were found to recover faster from EAE-associated symptoms than wild-type mice, encouraging the relevance also of the hCD83 as a key molecule for the regulatory phenotype of Tregs in vitro and in vivo.

CD83 expression is essential for Treg cell differentiation and stability.

Foxp3-positive regulatory T cells (Tregs) are crucial for the maintenance of immune homeostasis and keep immune responses in check. Upon activation, Tregs are transferred into an effector state expressing transcripts essential for their suppressive activity, migration, and survival. However, it is not completely understood how different intrinsic and environmental factors control differentiation. Here, we present for the first time to our knowledge data suggesting that Treg-intrinsic expression of CD83 is essential for Treg differentiation upon activation. Interestingly, mice with Treg-intrinsic CD83 deficiency are characterized by a proinflammatory phenotype. Furthermore, the loss of CD83 expression by Tregs leads to the downregulation of Treg-specific differentiation markers and the induction of an inflammatory profile. In addition, Treg-specific conditional knockout mice showed aggravated autoimmunity and an impaired resolution of inflammation. Altogether, our results show that CD83 expression in Tregs is an essential factor for the development and function of effector Tregs upon activation. Since Tregs play a crucial role in the maintenance of immune tolerance and thus prevention of autoimmune disorders, our findings are also clinically relevant.

Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo.

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations.

Determination of beta-carboline alkaloids in fruiting bodies of Hygrophorus spp. by liquid chromatography/electrospray ionisation tandem mass spectrometry.

The beta-carboline alkaloids harmane (1) and norharmane (2) were isolated from fruiting bodies of Hygrophorus eburneus (Bull.) Fr. as well as brunnein A (3) from Hygrophorus hyacinthinus Quél. (Tricholomataceae, Agaricales) for the first time. Their occurrence within the genus was investigated using liquid chromatography/electrospray ionisation tandem mass spectrometric methods, especially by selected reaction monitoring. Based on these results their chemotaxonomical relevance is discussed.

Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding.

Papaver alkaloids play a major role in medicine and pharmacy. In this study, [ring-(13)C(6)]-tyramine as a biogenetic precursor of these alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. Thus, based on this procedure, the structure of about 20 alkaloids displaying an incorporation of the labeled tyramine could be elucidated. These alkaloids belong to different classes, e.g. morphinan, benzylisoquinoline, protoberberine, benzo[c]phenanthridine, phthalide isoquinoline and protopine. The valuable information gained from the alkaloid profile demonstrates that the combination of these two spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products.

Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments.

The coumarin antibiotic coumermycin A(1) contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A(1) biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A(1) production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A(1), respectively. C-methylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the non-methylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A(1) in the wild-type.

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis.

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.