Increased Development of Th1, Th17, and Th1.17 Cells Under T1 Polarizing Conditions in Juvenile Idiopathic Arthritis.

In juvenile idiopathic arthritis (JIA) inflammatory T cells and their produced cytokines are drug targets and play a role in disease pathogenesis. Despite their clinical importance, the sources and types of inflammatory T cells involved remain unclear. T cells respond to polarizing factors to initiate types of immunity to fight infections, which include immunity types 1 (T1), 2 (T2), and 3 (T17). Polarizing factors drive CD4+ T cells towards T helper (Th) cell subtypes and CD8+ T cells towards cytotoxic T cell (Tc) subtypes. T1 and T17 polarization are associated with autoimmunity and production of the cytokines IFNγ and IL-17 respectively. We show that JIA and child healthy control (HC) peripheral blood mononuclear cells are remarkably similar, with the same frequencies of CD4+ and CD8+ naïve and memory T cell subsets, T cell proliferation, and CD4+ and CD8+ T cell subsets upon T1, T2, and T17 polarization. Yet, under T1 polarizing conditions JIA cells produced increased IFNγ and inappropriately produced IL-17. Under T17 polarizing conditions JIA T cells produced increased IL-17. Gene expression of IFNγ, IL-17, Tbet, and RORγT by quantitative PCR and RNA sequencing revealed activation of immune responses and inappropriate activation of IL-17 signaling pathways in JIA polarized T1 cells. The polarized JIA T1 cells were comprised of Th and Tc cells, with Th cells producing IFNγ (Th1), IL-17 (Th17), and both IFNγ-IL-17 (Th1.17) and Tc cells producing IFNγ (Tc1). The JIA polarized CD4+ T1 cells expressed both Tbet and RORγT, with higher expression of the transcription factors associated with higher frequency of IL-17 producing cells. T1 polarized naïve CD4+ cells from JIA also produced more IFNγ and more IL-17 than HC. We show that in JIA T1 polarization inappropriately generates Th1, Th17, and Th1.17 cells. Our data provides a tool for studying the development of heterogeneous inflammatory T cells in JIA under T1 polarizing conditions and for identifying pathogenic immune cells that are important as drug targets and diagnostic markers.

A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates.

We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.

Bromodomain inhibitor JQ1 reversibly blocks IFN-γ production.

As a class, 'BET' inhibitors disrupt binding of bromodomain and extra-terminal motif (BET) proteins, BRD2, BRD3, BRD4 and BRDT, to acetylated histones preventing recruitment of RNA polymerase 2 to enhancers and promoters, especially super-enhancers, to inhibit gene transcription. As such, BET inhibitors may be useful therapeutics for treatment of cancer and inflammatory disease. For example, the small molecule BET inhibitor, JQ1, selectively represses MYC, an important oncogene regulated by a super-enhancer. IFN-γ, a critical cytokine for both innate and adaptive immune responses, is also regulated by a super-enhancer. Here, we show that JQ1 represses IFN-γ expression in TH1 polarized PBMC cultures, CD4+ memory T cells, and NK cells. JQ1 treatment does not reduce activating chromatin marks at the IFNG locus, but displaces RNA polymerase II from the locus. Further, IFN-γ expression recovers in polarized TH1 cultures following removal of JQ1. Our results show that JQ1 abrogates IFN-γ expression, but repression is reversible. Thus, BET inhibitors may disrupt the normal functions of the innate and adaptive immune response.