Abstract
Purpose: :
Persistent production of TNF occurs in many autoimmune inflammatory diseases, including uveitis, and can lead to significant tissue damage. Several autoimmune diseases are treated by targeting TNF with neutralising antibodies and soluble TNF receptors but not everyone benefits. Therefore, there are opportunities for new treatments. High serum concentrations of TNF, IL-1β, IL-6 and IL-8 have been detected in patients with inflammatory autoimmune diseases; while in contrast, the levels of IL-4, IL-10 and TGFβ are reduced. This suggests indirectly that failure to produce an appropriate balance of inhibitory factors contributes to the pathogenesis of inflammatory autoimmune diseases. One mechanism of TNF regulation is via control of mRNA stability. TGFβ1 may mediate its inhibitory effects via TNF mRNA stability, but this remains to be demonstrated. The TNF mRNA 3’ untranslated region (UTR) contains an AU-rich element (ARE), which targets mRNAs for degradation. RNA-binding proteins (i.e. TTP, TIA-1, TIAR, and FXR1) have been shown to bind the TNF ARE. We have studied the role of TGFβ1 in the induction of some of these RNA binding proteins. Understanding the homeostatic control of TNF by TGFβ1 further will eventually allow for more effective therapies.
Methods: :
Using RAW 264.7 cells and mouse bone-marrow derived macrophages stimulated with LPS and TGFβ1, we assessed mRNA expression by Q-PCR and protein expression by flow cytometry using an intracellular stain for TNF. We studied the effects of TGFβ1 on LPS-induced TNF mRNA decay using actinomycin D, a transcription inhibitor, followed by Q-PCR. We isolated the 3’UTR of TNF mRNA and inserted it into a luciferase reporter vector on a constitutive promoter. This plasmid was transfected into RAW cells that were treated with LPS and TGFβ1. We studied the effects of TGFβ1 and LPS on FXR1 expression using Q-PCR, Western Blot and siRNA knockdown.
Results: :
We find that while TNF mRNA expression stays fairly constant, TGFβ1 inhibits LPS induced TNF protein expression. Using actinomycin D to study mRNA stability, we show that TGFβ1 decreases the half-life of TNF mRNA which is increased by LPS. Using the luciferase-TNF-3’UTR vector we show that TGFβ1 affects the 3’UTR of TNF. These effects may be mediated via FXR1.
Conclusions: :
Macrophages constitutively express TNF mRNA, which is unstable and therefore they do not produce TNF protein. LPS stabilises TNF mRNA, leading to an increase in TNF protein and TGFβ1 destabilises TNF mRNA via the 3’UTR, therefore reducing the amount of TNF protein. These effects involve FXR1.
Keywords: autoimmune disease • cytokines/chemokines • inflammation