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It has been suggested that glucocorticoid receptor (GR) agonists that promote GR homodimerization more than standard glucocorticoids such as Dexamethasone could be more effective anti-inflammatory molecules against acute and life-threatening inflammatory conditions. To test this hypothesis, we set up a screening pipeline aimed at discovering such Selective Dimerizing GR Agonists and Modulators (SEDIGRAM). The pipeline consists of a reporter gene assay based on a palindromic glucocorticoid responsive element (GRE). This assay represents GR dimerization in human A549 lung epithelial cells. In the pipeline, this is followed by analysis of endogenous GRE-driven gene expression, a FRET assay confirming dimerization, and monitoring of in vitro and in vivo anti-inflammatory activity. In a proof of principle experiment, starting from seven candidate compounds, we identified two potentially interesting compounds (Cortivazol and AZD2906) that confer strong protection in a mouse model of aggressive TNF-induced lethal inflammation. A screening pipeline for SEDIGRAM may assist the search for compounds that promote GR dimerization and limit overwhelming acute inflammatory responses.

TNF has as detrimental role in multiple sclerosis (MS), however, anti-TNF medication is not working. Selective TNF/TNFR1 inhibition whilst sparing TNFR2 signaling reduces the pro-inflammatory effects of TNF but preserves the important neuroprotective signals via TNFR2. We previously reported the generation of a Nanobody-based selective inhibitor of human TNFR1, TROS that will be tested in experimental autoimmune encephalomyelitis (EAE). We specifically antagonized TNF/TNFR1 signaling using TROS in a murine model of MS, namely MOG35-55-induced EAE. Because TROS does not cross-react with mouse TNFR1, we generated mice expressing human TNFR1 in a mouse TNFR1-knockout background (hTNFR1 Tg), and we determined biodistribution of 99mTc-TROS and effectiveness of TROS in EAE in those mice. Biodistribution analysis demonstrated that intraperitoneally injected TROS is retained more in organs of hTNFR1 Tg mice compared to wild type mice. TROS was also detected in the cerebrospinal fluid (CSF) of hTNFR1 Tg mice. Prophylactic TROS administration significantly delayed disease onset and ameliorated its symptoms. Moreover, treatment initiated early after disease onset prevented further disease development. TROS reduced spinal cord inflammation and neuroinflammation, and preserved myelin and neurons. Collectively, our data illustrate that TNFR1 is a promising therapeutic target in MS.

Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.