GPA is a type of anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, an autoimmune disease caused by the production of autoantibodies — antibodies that wrongly target and attack healthy cells. This leads to blood vessel inflammation, and swelling in affected tissues and organs.
As in other types of autoimmune diseases, regulatory T-cells (Tregs) — immune cells that regulate the function of B-cells and other types of T-cells — often malfunction in GPA patients.
“In vitro experiments have shown that circulating Tregs from GPA patients have a reduced ability to suppress the proliferation of activated effector cells [killer immune cells]. However, the exact mechanisms that contribute to the functional impairment of Tregs in GPA are currently unknown,” the investigators said.
MicroRNAs (miRNAs) — tiny RNA molecules that control the expression of several genes — have recently been implicated in the regulation of immune T-cells’ function.
“To date, it is unknown whether miRNAs are differentially expressed in Tregs of GPA patients and whether specific miRNAs are linked to the observed impaired suppressive function of these Tregs,” the researchers said.
Investigators from the University of Groningen in the Netherlands now set out to explore if changes in the levels of certain miRNAs could be responsible for the dysregulation of Tregs often seen in people with GPA.
To investigate that hypothesis, they isolated RNA — the molecule that serves as a template for the production of proteins — from Tregs of eight untreated GPA patients and eight healthy individuals (controls).
Then, to look for miRNAs that could be present in abnormal levels in Tregs from GPA, they performed a microarray analysis, which is a technique scientists use to measure the expression levels of genes.
The microarray analysis revealed there were 19 miRNAs whose levels were either too high or too low in Tregs obtained from GPA patients compared with those isolated from healthy individuals.
Among these 19 miRNAs, miR-142-3p was one of those whose levels were significantly higher — almost two-fold — in Tregs from people with GPA compared with controls.
When investigators forced Tregs from healthy individuals to produce large amounts of miR-142-3p, they found these cells lost their ability to suppress the growth of other T-cells. Moreover, correlation analysis confirmed the levels of miR-142-3p were inversely correlated with Tregs’ suppressive capacity.
Adenylate cyclase 9 (ADCY9) is a potential target of miR-142-3p. An enzyme responsible for the production of cyclic adenosine monophosphate (cAMP), ADCY9 is necessary for Tregs to be able to control growth of other T-cells.
To see if miR-142-3p could affect the production of ADCY9 and cAMP in Tregs, investigators measured the levels of both molecules in Tregs from healthy individuals who were forced to produce large amounts of miR-142-3p. Their experiments revealed that Tregs containing high levels of miR-142-3p produced less cAMP and ADCY9 messenger RNA (mRNA).
In addition, when investigators measured the levels of cAMP and ADCY9 in Tregs from GPA patients, they found these cells produced two times less ADCY9 mRNA and significantly less cAMP compared with controls. This was in agreement with their previous findings.
Finally, they showed the negative effects of miR-142-3p on Tregs’ suppressive ability could be reversed by treating cells with forskolin, an agent that boosts the production of cAMP.
“Our results suggest that therapeutic interventions aiming to restore miR-142-3p and cAMP levels in Tregs present a novel approach to restore Treg function in GPA patients and potentially in those with other autoimmune diseases in which there is a functional defect in the Treg subset,” the researchers said.