Increased miR-142-3p Expression Might Explain Reduced Regulatory T Cell Function in Granulomatosis With Polyangiitis

Abstract

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression. Methods: RNA isolated from FACS-sorted memory (_M) Tregs (CD4⁺CD45RO⁺CD25⁺CD127^-) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in _MTregs from patients and HCs. cAMP levels were measured using flow cytometry. Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in _MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of _MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, _MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed _MTregs by forskolin restored their suppressive function in vitro. Conclusion: Overexpression of miR-142-3p in _MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

Keywords: ANCA—associated vasculitis; Treg—regulatory T cell; auto immune; microRNA; suppressive function.

Figure 1

Frequencies of CD4⁺T cells and CD4⁺T cell subsets in healthy controls (HC) and GPA patients in remission (GPA). Representative FACS plots of sorted CD4⁺T cell subsets (A). CD4⁺T cell frequencies were lower in GPA than in HC (B). Within the CD4⁺T cell population, no differences were observed in naïve T (T_NAVE) cells (CD4⁺CD45RO⁻CCR7⁻) (C), whereas the _MTreg (CD4⁺CD127⁻CD25^HighCD45RO⁺) frequency was higher in GPA than in HC (D). A significantly higher frequency of CD4⁺T effector memory (T_EM) cells (CD4⁺CD45RO⁺CCR7⁻) was observed in GPA than in HC (E). **P = 0.01–0.001, ***P ≤ 0.001.

Figure 2

miR-142-3p is overexpressed in _MTregs from GPA patients in remission and is differentially regulated in CD4⁺T cell subsets. miR-142-3p levels were significantly higher in _MTregs from GPA patients (GPA) than in those from healthy controls (HC) (A). miR-142-3p levels were significantly lower in _MTregs than in _NTregs (B) and in CD4⁺T_NAVE and CD4⁺T_EM cells from HC (circles) and GPA patients (squares) (C). Stimulation of _MTregs and _NTregs with anti-CD3-CD28 Dynabeads led to significantly lower miR-142-3p levels over time (n = 5) (D). *P = 0.05–0.01, **P = 0.01–0.001.

Figure 3

Overexpression of miR-142-3p reduces the suppressive capacity of _MTregs in vitro. _MTregs were transfected with scrambled (SCR) or miR-142-3p mimic (MIM-142-3p). Live _MTregs were sorted and cocultured with responder T (T_RESP) cells; after 48 h of stimulation, T_RESP cell proliferation was determined (A,B). Transfection with MIM-142-3p significantly increased miR-142-3p levels (C) and reduced the suppressive capacity of _MTregs (D). miR-142-3p levels correlated negatively with the suppressive capacity (E). Transfection with MIM-142-3p further decreased ADCY9 mRNA levels (F) and correlated negatively with miR-142-3p levels (G). Additionally, cAMP levels were significantly lower in MIM-142-3p-transfected _MTregs (red) than in SCR-transfected _MTregs (black) (H). *p = 0.05–0.01.

Figure 4

_MTreg ADCY9 mRNA and cAMP levels are reduced in patients with GPA. ADCY9 mRNA levels were significantly lower in patients in remission (GPA) (A), but mRNA levels of ADCY9 did not correlate with miR-142-3p expression (B). In the cAMP cohort, cAMP levels were measured in _MTregs upon stimulation. cAMP levels in _MTregs were higher in healthy controls (HC) after 48 h stimulation (n = 10) than in GPA (n = 10) (C), and total cAMP production was significantly higher in HC than in GPA (D). Besides cAMP, FoxP3 expression was also higher in HC after stimulation (E) and FoxP3 levels correlated with cAMP (F). *p = 0.05–0.01, **p = 0.01–0.001.

Figure 5

cAMP elevating agent Forskolin increases Treg function via the increase of cAMP. As proof of principle we tried to restore Treg function after miR-142-3p overexpression using cAMP elevating agent Forskolin. _MTregs were transfected with scrambled (SCR) or miR-142-3p mimic (MIM-142-3p). Live _MTregs were sorted and cocultured with responder T (T_RESP) cells; after 48 h of stimulation, T_RESP cell proliferation was determined (A). Upon treatment with Forskolin, cAMP levels increased significantly (B). Moreover, forskolin treatment tended to restore suppressive function in miR-142-3p overexpressed _MTregs (C).*p = 0.05–0.01, **p = 0.01–0.001.

Figure 6

Proposed model of miR-142-3p, which targets the adenylyl cyclase 9/cAMP-mediated suppression of Tregs. We propose that miR-142-3p overexpression in GPA patients reduces ADCY9 levels, which leads to less conversion of ATP to cAMP upon stimulation. cAMP can be transferred to effector cells via a Gap junction (GJ). Upon transfection, cAMP levels in effector cells increase, which induces metabolic disruption and inhibits IL-2 production and cell proliferation. Increased miR-142-3p levels therefore decrease Treg function.