Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb 13;17(1):32.
doi: 10.1186/s13075-015-0548-y.

Estrogen regulates T helper 17 phenotype and localization in experimental autoimmune arthritis

Annica Andersson  1 , Alexandra Stubelius  2 , Merja Nurkkala Karlsson  3 , Cecilia Engdahl  4   5 , Malin Erlandsson  6 , Louise Grahnemo  7 , Marie K Lagerquist  8 , Ulrika Islander  9
Affiliations

Estrogen regulates T helper 17 phenotype and localization in experimental autoimmune arthritis

Annica Andersson et al. Arthritis Res Ther. .

Abstract

Introduction: The incidence and progression of many autoimmune diseases are sex-biased, which might be explained by the immunomodulating properties of endocrine hormones. Treatment with estradiol potently inhibits experimental autoimmune arthritis. Interleukin-17-producing T helper cells (Th17) are key players in several autoimmune diseases, particularly in rheumatoid arthritis. The aim of this study was to investigate the effects of estrogen on Th17 cells in experimental arthritis.

Methods: Ovariectomized DBA/1 mice treated with 17β-estradiol (E2) or placebo were subjected to collagen-induced arthritis (CIA), and arthritis development was assessed. Th17 cells in joints and lymph nodes were studied by flow cytometry. Lymph node Th17 cells were also examined in ovariectomized estrogen receptor α-knockout mice (ERα-/-) and wild-type littermates, treated with E2 or placebo and subjected to antigen-induced arthritis.

Results: E2-treated mice with established CIA showed reduced severity of arthritis and fewer Th17 cells in joints compared with controls. Interestingly, E2-treated mice displayed increased Th17 cells in lymph nodes during the early phase of the disease, dependent on ERα. E2 increased the expression of C-C chemokine receptor 6 (CCR6) on lymph node Th17 cells as well as the expression of the corresponding C-C chemokine ligand 20 (CCL20) within lymph nodes.

Conclusions: This is the first study in which the effects of E2 on Th17 cells have been characterized in experimental autoimmune arthritis. We report that E2 treatment results in an increase of Th17 cells in lymph nodes during the early phase of arthritis development, but leads to a decrease of Th17 in joints during established arthritis. Our data suggest that this may be caused by interference with the CCR6-CCL20 pathway, which is important for Th17 cell migration. This study contributes to the understanding of the role of estrogen in the development of autoimmune arthritis and opens up new fields for research concerning the sex bias in autoimmune disease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Experimental setup for collagen-induced arthritis. Female DBA/1 mice were ovariectomized (OVX) at 8 to 10 weeks of age and simultaneously received an implanted slow-release pellet containing 17β-estradiol (E2; 0.83 μg/day) or corresponding placebo. After 12 days of rest, they were immunized subcutaneously at the base of the tail with collagen type II and Freund’s adjuvant (day 0), which was repeated at day 21 or 28. Experiments were terminated at days 14, 23, 35 and 49 after the first immunization. Days 14 and 23 were considered as time points in the (asymptomatic) induction phase of collagen-induced arthritis (CIA), whereas days 35 and 49 were defined as established (symptomatic) CIA.
Figure 2
Figure 2
Effects of estrogen on arthritis development in the collagen-induced arthritis model. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. Arthritis development was macroscopically assessed every second or third day by a blinded examiner, yielding a maximum of 12 points per mouse (single experiment with n = 14 mice/group). (A) Severity of arthritis is expressed as median and interquartile range. The area under the curve was calculated for each treatment group and differences between groups were analyzed by Mann–Whitney test. CII, Collagen type II. (B) Incidence of arthritis is presented as Kaplan-Meier curves and analyzed by log-rank test. (C) Microscopic synovitis and erosions on bone and cartilage were assessed in paw sections stained with hematoxylin and eosin, from placebo- or E2-treated mice with CIA, terminated on day 49. Data are median with interquartile range, Mann–Whitney test, n = 5 or 6 mice/group in a single experiment. Representative sections from each treatment group are shown in (D). Original magnification, ×20. *P <0.05, ***P <0.001.
Figure 3
Figure 3
Effects of estrogen on Th17 cells in joints of mice with established collagen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. (A) Joint cells from mice with CIA terminated day 49 were stimulated in triplicates with phorbol 12-myristate 13-acetate and ionomycin for 40 to 42 hours in interleukin (IL)-17 ELISPOT assay. The number of spot-forming cells (SFCs) was counted in a blinded manner these are shown as SFCs per total number of cells in wells. Total numbers of (B) T helper 17 cells (Th17; IL-17+CD4+) and (C) neutrophils (CD11b+F4/80−Gr-1hi) in joints from mice with CIA terminated day 35 were determined by flow cytometry. The absolute number of cells was derived by multiplying the frequency of Th17 cells or neutrophils by the total number of cells acquired from an automated cell counter in a corresponding sample. All data shown are arithmetic mean ± SEM derived from single experiments with n = 8 or 9 mice/group. *P <0.05 (Student’s t-test).
Figure 4
Figure 4
Estrogenic effects on lymph node Th17 cells in collagen-induced arthritis and in antigen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) (A–E), or ovariectomized estrogen receptor α–knockout mice (ERα−/−) mice and wild-type (WT) littermates were subjected to antigen-induced arthritis (AIA) (F) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo (A, B). The frequency of T helper 17 (Th17) cells in lymph nodes (LNs) of CIA mice (terminated at day 14, n = 10 mice/group; at day 23, n = 8 to 10 mice/group; at day 35, n = 7 to 9 mice/group). Data from day 14 is from one representative experiment out of three independent experiments in total; otherwise, single experiments were performed. Representative flow cytometry plots are shown in (A). (C) Frequency of Th17 cells in LNs of CIA mice (day 23, single experiment, n = 9 mice/group), with only 3 days of E2 treatment (days 20 to 22). (D , E) Frequencies of Th1 (D) and regulatory T cells (Treg) (E) in LNs of CIA mice (day 14, n = 10 mice/group; day 23, n = 8 to 10 mice/group; single experiments). (F) In AIA, frequency of Th17 cells in LNs (draining knee joints) was determined at day 14 (two independent experiments, in total n = 14 to 19 mice/group). To improve normal distribution of data, some data were log-transformed prior to statistical analysis (B, D and E). Arithmetic mean ± SEM is shown in all graphs. Student’s t-test was used (*P <0.05, **P <0.01).
Figure 5
Figure 5
Absolute numbers of lymph node CD4 + cells and Th17 cells after estrogen treatment in early collagen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. (A,B) Absolute number of T helper 17 (Th17) cells (A) and CD4+ cells (B) in one subiliac lymph node (LN) of CIA mice (day 14, n = 10 or 11 mice/group), obtained by multiplying cell population frequencies obtained by fluorescence-activated cell sorting by total LN cellularity. To improve normal distribution of data, some data were log-transformed prior to statistical analysis (A). Arithmetic mean ± SEM is shown in all graphs. Student’s t-test (B) or Welch’s t-test (A) was used (*P <0.05).
Figure 6
Figure 6
Th17 chemokine receptor expression and corresponding lymph node chemokine expression after estrogen treatment in collagen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. (A–C) Frequency of C-C chemokine receptor 2-positive (CCR2+) cells of T helper 17 (Th17) cells (A) and median fluorescence intensity (MFI) of CCR2 on Th17 cells (B,C) in lymph nodes (LNs) from CIA mice (day 14, single experiment with n = 10 or 11 mice/group). (B) Representative fluorescence-activated cell sorting (FACS) analysis plot where fluorescence minus one (FMO) is control. (D,E) mRNA expression of Ccl2 (D) and Ccl12 (E) in LNs of CIA mice (day 14, single experiment, n = 9 or 10 mice/group), determined by real-time quantitative PCR. (F–H) Frequency of CCR6+ cells of Th17 (F) and MFI of CCR6 on Th17 cells (G,H) in LNs of CIA mice (day 14, single experiments, n = 9 or 10 mice/group). (G) Representative FACS plot where FMO is control. (I) mRNA expression of Ccl20 in LNs of CIA mice (day 14, data pooled from two experiments, n = 20 mice/group). (J) CCL20-specific migration of interleukin (IL)-17+ cells. LN cells from mice with CIA (day 35) were put on a transwell assay, with CCL20 in the lower chamber, and the frequency of migrated cells was evaluated with an IL-17 ELISPOT assay. Data are representative of two independent experiments (n = 3 mice/group). To improve normal distribution of data, some data were log-transformed prior to statistical analysis (A, C, F and H). Bars or lines show arithmetic mean ± SEM (A, C, F, H and J) or geometric mean (D, E and I). Student’s t-test (A, C–F, H and J) or analysis of covariance with experiment as covariate (I) was used (*P <0.05, **P <0.01).
Figure 7
Figure 7
Th17 S1PR1 expression in lymph nodes in early collagen-induced arthritis. DBA/1 mice were ovariectomized, subjected to collagen-induced arthritis (CIA) and treated with 17β-estradiol (E2; 0.83 μg/day) or placebo. (A–C) Frequency of sphingosine-1-phosphate receptor 1-positive (S1PR1+) cells of T helper 17 (Th17) (A) and median fluorescence intensity (MFI) of S1PR1 on Th17 cells (B,C) in lymph nodes (LNs) from CIA mice (day 14, single experiment with n = 10 mice/group). (B) Representative fluorescence-activated cell sorting analysis plot where fluorescence minus one is used as a control. (D) S1P levels in serum of mice with CIA at day 14 were determined by enzyme-linked immunosorbent assay (single experiment, n = 9 or 10 mice/group). To improve normal distribution of data, some data were log-transformed prior to statistical analysis (A,C). Bars or lines show arithmetic mean ± SEM. Student’s t-test (**P <0.01).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Goemaere S, Ackerman C, Goethals K, De Keyser F, Van der Straeten C, Verbruggen G, et al. Onset of symptoms of rheumatoid arthritis in relation to age, sex and menopausal transition. J Rheumatol. 1990;17:1620–2. - PubMed
    1. Khosla S, Melton LJ, 3rd, Atkinson EJ, O’Fallon WM. Relationship of serum sex steroid levels to longitudinal changes in bone density in young versus elderly men. J Clin Endocrinol Metab. 2001;86:3555–61. doi: 10.1210/jcem.86.8.7736. - DOI - PubMed
    1. Ostensen M. Sex hormones and pregnancy in rheumatoid arthritis and systemic lupus erythematosus. Ann N Y Acad Sci. 1999;876:131–43. doi: 10.1111/j.1749-6632.1999.tb07630.x. - DOI - PubMed
    1. Holmdahl R, Carlsten H, Jansson L, Larsson P. Oestrogen is a potent immunomodulator of murine experimental rheumatoid disease. Br J Rheumatol. 1989;28:54–8. doi: 10.1093/rheumatology/XXVIII.suppl_1.54. - DOI - PubMed
    1. Engdahl C, Jochems C, Windahl SH, Börjesson AE, Ohlsson C, Carlsten H, et al. Amelioration of collagen-induced arthritis and immune-associated bone loss through signaling via estrogen receptor α, and not estrogen receptor β or G protein-coupled receptor 30. Arthritis Rheum. 2010;62:524–33. - PubMed

Publication types

LinkOut - more resources

-