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.

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

Autophagic degradation of lamins facilitates the nuclear egress of herpes simplex virus type 1.

Dendritic cells (DCs) are crucial for the induction of potent antiviral immune responses. In contrast to immature DCs (iDCs), mature DCs (mDCs) are not permissive for infection with herpes simplex virus type 1 (HSV-1). Here, we demonstrate that HSV-1 infection of iDCs and mDCs induces autophagy, which promotes the degradation of lamin A/C, B1, and B2 in iDCs only. This in turn facilitates the nuclear egress of progeny viral capsids and thus the formation of new infectious particles. In contrast, lamin protein levels remain stable in HSV-1-infected mDCs due to an inefficient autophagic flux. Elevated protein levels of KIF1B and KIF2A in mDCs inhibited lamin degradation, likely by hampering autophagosome-lysosome fusion. Therefore, in mDCs, fewer progeny capsids were released from the nuclei into the cytosol, and fewer infectious virions were assembled. We hypothesize that inhibition of autophagic lamin degradation in mDCs represents a very powerful cellular counterstrike to inhibit the production of progeny virus and thus viral spread.

A new promising candidate to overcome drug resistant herpes simplex virus infections.

Infections with Herpes simplex viruses (HSV) belong to the most common human diseases worldwide, resulting in symptoms ranging from painful, but commonly self-limiting lesions of the orofacial or genital tract to severe infections of the eye or life-threatening generalized infections. Frequent HSV-reactivations at the eye may lead to the development of herpetic stromal keratitis, which is one of the major causes of infectious blindness in developed countries. The vast majority of life-threatening generalized infections occur in immunocompromised individuals, such as transplant recipients or patients suffering from advanced human immunodeficiency virus (HIV) infection with concurrent HSV-reactivation. Over the past decades, Acyclovir (ACV) became the golden standard for the treatment of HSV infections. However, long-term antiviral treatment, as it is required mainly in immunocompromised patients, led to the emergence of resistances towards ACV and other antivirals. Therefore, there is a clear need for the development of new potent antivirals which combine good oral bioavailability and tolerability with low side effects. In the current study we present SC93305 as a novel potent antiviral substance that proved to be highly effective not only against different HSV-1 and HSV-2 strains but also towards ACV- and multi-resistant HSV-1 and HSV-2 isolates. SC93305 shows comparable antiviral activity as reported for ACV and very importantly it does not interfere with the activation of specific immune cells. Here we report that SC93305 does not affect the biological function of dendritic cells (DC), the most potent antigen presenting cells of the immune system to induce antiviral immune responses, nor T cell stimulation or the release of inflammatory cytokines. Thus, SC93305 is a new and promising candidate for the treatment of HSV-1 and HSV-2 infections and in particular also for the inhibition of drug-resistant HSV-1/2 strains.

Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration.

As potent antigen-presenting cells, dendritic cells (DCs) are essential for the initiation of effective antiviral immune responses. Viruses and especially herpesviruses, which are able to establish lifelong persistence, exploit several immune evasion mechanisms targeting DC biology. Our group has previously shown that the α-herpesvirus herpes simplex virus type 1 inhibits mature DC (mDC) migration by inducing adhesion via degrading the cellular protein CYTIP (cytohesin-1 interacting protein), an important negative regulator of ß2-integrin activity. In the present study, we extended our analysis to the ß-herpesvirus human cytomegalovirus (HCMV), to investigate whether other herpesviridae also induce such modulations. Indeed, HCMV impairs mDC transwell migration capability following a CCL19-chemokine gradient, despite equivalent expression levels of the cognate chemokine receptor CCR7 at the corresponding time points post-infection. Remarkably, HCMV infection potently induced ß2-integrin activity on mDCs. Furthermore, directly HCMV-infected mDCs, exhibiting viral gene expression, strongly adhere to fibronectin and ICAM-1, in contrast to mDCs lacking infection or viral gene expression. Interestingly, HCMV-positive mDCs display a proteasome-dependent degradation of CYTIP. Contrasting the migration toward CCL19, elevated expression levels of the chemokine receptor CXCR4 in HCMV-infected mDCs were associated with functional CXCL12-chemotaxis under the herein used conditions. In summary, our results show that HCMV shapes mDC adhesion to compromise migration toward CCL19, but retaining CXCL12 responsiveness. Thus, we hypothesize that a preferred migration pattern toward the bone marrow, but not to secondary lymphoid organs, could ultimately cause a failure in the induction of potent antiviral immune responses.

L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation.

UNLABELLED: Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE: HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.