Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis
Susanna Choi, … , Chul-Soo Cho, Wan-Uk Kim
Susanna Choi, … , Chul-Soo Cho, Wan-Uk Kim
Published February 13, 2017
Citation Information: J Clin Invest. 2017;127(3):954-969. https://doi.org/10.1172/JCI87880.
View: Text | PDF
Research Article Immunology Inflammation

Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis

Abstract

Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5+/– mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages.

Authors

Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim

×

Figure 6

The NFAT5 target CCL2 prevents apoptotic death of RA macrophages.

Options: View larger image (or click on image) Download as PowerPoint
The NFAT5 target CCL2 prevents apoptotic death of RA macrophages. (A) mR...
(A) mRNA expression levels of representative NFAT5-regulated genes involved in cell survival, proliferation, and apoptosis in RA-SF macrophages (n = 8) compared with normal macrophages (n = 8), as determined by real-time PCR and presented according to fold change. (B and C) Increased CCL2 expression in CD14+ cells in response to NFAT5-inducing stimuli. Peripheral blood CD14+ cells (1 × 106 cells) from healthy controls (n = 6) were stimulated with M-CSF (20 ng/ml), IL-1β (10 ng/ml), and LPS (100 ng/ml) for 12 hours (B) or 24 hours (C). CCL2 expression in the culture supernatants was determined by ELISA (B) and intracellular flow cytometry (C). (D) NFAT5 expression in CCL2+ versus CCL2– subpopulations of RA-SF macrophages (n = 3), as determined by flow cytometry. Gates for the CCL2+ or CCL2– cells are shown in the flow cytometric dot plot. Histogram shows the difference in NFAT5 expression according to CCL2 positivity. MFI, mean fluorescence intensity. (E) Decreased CCL2 secretion by NFAT5-deficient human macrophages. Peripheral CD14+ cells (1 × 106 cells, n = 4) were stimulated with M-CSF for 24 hours and then transfected with NFAT5 shRNA or control shRNA for a further 24 hours. CCL2 levels in the culture supernatants were measured by ELISA (bar graph) and were also assessed by flow cytometry (flow cytometric dot plot) in macrophages 24 hours after stimulation with 10 ng/ml LPS. (F) Effect of CCL2 on NFAT5-dependent apoptosis of RA-SF macrophages. RA-SF macrophages (2 × 105 cells) were transfected with NFAT5 shRNA or control shRNA for 24 hours. The cells were then cultured in the presence or absence of 100 ng/ml recombinant CCL2 (rCCL2) (n = 5) for 24 hours. The extent of apoptosis was determined by flow cytometry for annexin V. Bar graphs in A–F represent the mean ± SD. (A) *P < 0.05 and **P < 0.001 versus normal macrophages, by Mann-Whitney U test. (B and C) **P < 0.001 versus media only, by 1-way ANOVA with Tukey’s post-hoc test. (D, E, and F) *P < 0.05 between groups, by Mann-Whitney U test. FSC, forward scatter; SSC, side scatter.