SUMMARY: Glucocorticoids (GCs) have been widely used for decades as a first-line treatment for inflammatory and autoimmune diseases. However, their use is often hampered by the onset of adverse effects or resistance. GCs mediate their effects via binding to glucocorticoid receptor (GR), a transcription factor belonging to the family of nuclear receptors. An important aspect of GR's actions, including its anti-inflammatory capacity, involves its interactions with various proteins, such as transcription factors, cofactors, and modifying enzymes, which codetermine receptor functionality. In this review, we provide a state-of-the-art overview of the protein-protein interactions (PPIs) of GR that positively or negatively affect its anti-inflammatory properties, along with mechanistic insights, if known. Emphasis is placed on the interactions that affect its anti-inflammatory effects in the presence of inflammatory and microbial diseases.

Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies^®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection.

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer–dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GR^WT/WT mice, these GR^dim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GR^dim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GR^dim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Natural variation for LPS‐induced lethal inflammation in mice is useful for identifying new genes that regulate sepsis, which could form the basis for novel therapies for systemic inflammation in humans. Here we report that LPS resistance of the inbred mouse strain SPRET/Ei, previously reported to depend on the glucocorticoid receptor (GR), maps to the distal region of the X‐chromosome. The GR‐inducible gene Tsc22d3 , encoding the protein Gilz and located in the critical region on the X‐chromosome, showed a higher expressed SPRET/Ei allele, regulated in cis . Higher Gilz levels were causally related to reduced inflammation, as shown with knockdown and overexpression studies in macrophages. Transient overexpression of Gilz by hydrodynamic plasmid injection confirmed that Gilz protects mice against endotoxemia Our data strongly suggest that Gilz is responsible for the LPS resistance of SPRET/Ei mice and that it could become a treatment option for sepsis.