In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

BackgroundImmunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity.ObjectivesWe used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging.MethodsWe assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects.ResultsOld human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase–related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003).ConclusionExcessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.

BackgroundPsoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy.Methods and FindingsThe preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin.ConclusionsOur work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.