Identification of novel inducers for LL-37 expression : assay development and mechanistic studies

Sammanfattning: The remaining reservoir of effective antibiotics is running out and more and more pathogens are gaining resistance, even to the last line of antibiotics. Therefore, novel treatment regimens against bacterial infections are urgently needed. Antimicrobial peptides (AMPs) are expressed at mucosal surfaces and protect the host against invasive microbes. Not only are they efficient microbial killers but they also orchestrate additional immune responses, including chemotaxis and wound healing. The focus in the current work has been on the human cathelicidin LL-37 (encoded by the CAMP gene), which can be considered as a marker for the AMP-system in general. Since several AMPs with different mechanism of action are released simultaneously, the risk of developing bacterial resistance is very low. Here we propose a concept where drug-like molecules that induce the expression of AMPs can be used to prevent or treat infections. We suggest that this novel approach designated ‘Host Directed Therapy’ can be used alone or together with traditional antibiotics for treatment of infections. The overall aim with this thesis was to identify novel AMP inducing compounds and to dissect the regulatory pathways involved in their mechanism of action. We set out to establish a cell-based reporter-gene-assay under the control of regulatory elements of the CAMP gene. Following a successful evaluation for high throughput screening purposes, six out of 1200 tested compounds were validated as novel inducers. One of these was the histone deacetylase (HDAC) inhibitor entinostat, which was found to be a potent inducer of LL-37 expression. Several derivatives of entinostat were synthesized and evaluated for LL-37 inducing activity. This strategy demonstrated which part of the molecule that was important for the induction. Entinostat was shown to regulate LL-37 via activation of the transcription factors STAT3 and HIF-1α. Notably, HIF-1α was found to directly bind and initiate transcription of the CAMP gene. In contrast, no direct binding of STAT3 to the CAMP gene promoter could be demonstrated. However, STAT3 was clearly involved in the regulation of LL-37 expression, since macrophages from a patient carrying a mutated STAT3 gene were unable to upregulate LL-37 upon entinostat treatment. Several additional regulatory pathways were discovered utilizing label free quantitative mass spectrometry, where protein levels were compared between unstimulated cells and cells treated with the AMP-inducing compounds lactose and phenylbutyrate. Next, the proteomic profiles were mapped to known pathways utilizing bioinformatics tools. The identified pathways were validated using the cell based reporter assay. The thyroid hormone receptor pathway, the eicosanoid signaling pathway and the steroid biosynthesis pathway could all be attributed to LL-37 expression. Together these results indicate that AMPs are strictly regulated both in homeostasis and during infection. The novel AMP-inducing compounds identified here should be further evaluated as Host Directed Therapy against infections in cellular and animal models as well as in relevant disease cohorts.

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