Accepted Manuscript Title: Selective estrogen receptor modulators in T cell development and T cell dependent inflammation Author: Angelina I. Bernardi Annica Andersson Alexandra Stubelius Louise Grahnemo Hans Carlsten Ulrika Islander PII: DOI: Reference:
S0171-2985(15)00077-7 http://dx.doi.org/doi:10.1016/j.imbio.2015.05.009 IMBIO 51322
To appear in: Received date: Revised date: Accepted date:
26-1-2015 26-2-2015 1-5-2015
Please cite this article as: Bernardi, A.I., Andersson, A.,Selective estrogen receptor modulators in T cell development and T cell dependent inflammation, Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.05.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short title: Effects of SERMs on T cells
Selective estrogen receptor modulators in T cell development and T cell dependent inflammation
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Angelina I. Bernardi, Annica Andersson, Alexandra Stubelius, Louise Grahnemo, Hans Carlsten, Ulrika Islander
Centre for Bone and Arthritis Research, Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, University of Gothenburg, Sweden
[email protected] [email protected] [email protected] [email protected] [email protected] [email protected]
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Angelina I. Bernardi, MSc: Annica Andersson, MSc: Alexandra Stubelius, PhD Louise Grahnemo, PhD: Hans Carlsten, MD, PhD, Professor: Ulrika Islander, PhD:
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Corresponding author: Angelina I. Bernardi Dept. of Rheumatology and Inflammation Research Sahlgrenska Academy, University of Gothenburg Box 480, 405 30 Gothenburg, Sweden Phone: +46 31 342 64 11 e-mail:
[email protected]
Keywords: Selective estrogen receptor modulators; estrogen; T cell development; delayed-
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type hypersensitivity
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Abbreviations: Las: lasofoxifene, bza: bazedoxifene, SERMs: selective estrogen receptor modulators, E2: 17-estradiol, ral: raloxifene, ovx: ovariectomized, DTH: delayed-type hypersensitivity, OXA: oxazolone, DN: double-negative, DP: double positive, SP: singlepositive, APCs: antigen-presenting cells, ER: estrogen receptor alpha, ER: estrogen receptor beta, SCID: severe combined immunodeficient, CIA: collagen-induced arthritis
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1. Abstract Lasofoxifene (las) and bazedoxifene (bza) are third generation selective estrogen receptor modulators (SERMs) with minimal estrogenic side effects, approved for treatment of
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postmenopausal osteoporosis. T cells are involved in the pathology of postmenopausal
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osteoporosis and previous studies have established an important role for 17-estradiol (E2) in T cell development and function. E2 causes a drastic thymic atrophy, alters the composition of thymic T cell populations, and inhibits T cell dependent inflammation. In contrast, the
second generation SERM raloxifene (ral) lacks these properties. Although las and bza are
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drugs approved for treatment of postmenopausal bone loss, it is of importance to study their effects on other biological aspects in order to extend the potential use of these compounds.
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Therefore, the aim of this study was to investigate if treatment with las and bza affects T lymphopoiesis and T cell dependent inflammation. C57Bl6 mice were ovariectomized (ovx)
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and treated with vehicle, E2, ral, las or bza. As expected, E2 reduced both thymus weight and decreased the proportion of early T cell progenitors while increasing more mature T cell
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populations in the thymus. E2 also suppressed the T cell dependent delayed-type hypersensitivity (DTH) reaction to oxazolone (OXA). Ral and las, but not bza, decreased
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thymus weight, while none of the SERMs had any effects on T cell populations in the thymus or on inflammation in DTH. In conclusion, this study shows that treatment with las or bza
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does not affect T lymphopoiesis or T cell dependent inflammation.
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2. Introduction Estrogen deficiency is associated with low bone mass and both human and experimental
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studies have shown that treatment with 17estradiol (E2) protects against bone loss [1].
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Selective estrogen receptor modulators (SERMs) are compounds that act as estrogen receptor
agonists or antagonists depending on the tissue. SERMs, exhibiting agonistic effects on bone, and antagonistic effects on reproductive organs, are efficient as treatment of postmenopausal osteoporosis. Raloxifene (ral), a second generation SERM, was the first SERM approved for treatment of postmenopausal osteoporosis. The new third generation SERMs lasofoxifene
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(las) and bazedoxifene (bza) have even better safety profiles and are indicated for treatment and prevention of postmenopausal osteoporosis [2]. It has been suggested that subclinical
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inflammation, with slightly elevated levels of pro-inflammatory cytokines, plays a role in
deficient osteoporosis [4].
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postmenopausal osteoporosis [3]. Moreover, T cells are critical in the pathology of estrogen-
E2 has diverse effects on the immune system such as inhibiting both T and B cell
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development, and stimulating antibody production [5-7]. It has repeatedly been shown that
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treatment with E2 improves disease development in experimental models of arthritis [8-10], and we have previously shown that the second generation SERM ral inhibits experimental
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postmenopausal arthritis [11]. Interestingly, preliminary data from our lab show that also las and bza have beneficial effects on disease development and associated bone loss in postmenopausal collagen-induced arthritis (Andersson et al, submitted). We recently reported that las and bza inhibit B lymphopoiesis at a later developmental stage compared to E2, and that they do not increase the number of antibody-producing cells or the marginal zone B cell population [12]. However, no previous studies have explored the effects of las and bza on T cell development.
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T lymphocytes develop in the thymus from lymphoid progenitor cells which migrate to the thymus from the bone marrow [13]. The cells initially lack both CD4 and CD8 and are termed double-negative (DN) T cells, which in turn can be divided in developmental order
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into DN1-DN4 subpopulations. DN4 cells subsequently acquire CD4 and CD8
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simultaneously and become double-positive (DP) T cells before differentiating into mature, either CD4+ or CD8+ single-positive (SP) T cells, which leave the thymus and enter the
periphery [14]. Removal of endogenously produced estrogen by ovariectomy (ovx) results in increased weight and cellularity of the thymus [15]. Phenotypical analysis of thymocytes after
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ovx have revealed that the increase in thymus cellularity mostly consists of a shift towards more DP cells and a decrease in DN and SP cells [15]. Conversely, in both animals and
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humans, the thymus involutes dramatically during pregnancy and after treatment with E2 [1618]. Histological studies of mouse thymus have shown that both pregnancy and treatment
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with E2 reduce the size of the cortex while the medulla is preserved, implying an E2-mediated loss of cortical thymocytes [19, 20]. In addition, treatment with E2 results in reduced thymic
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T cell number and a reduced proportion of DP cells, but increased levels of SP cells and DN1 cells, while the DN2-DN4 populations decrease[21], [22], [23]. However, treatment with the
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second generation SERM ral only induces a minor thymic atrophy, and in contrast to E2, ral does not alter the proportions of DP or SP T cells in the thymus [24].
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Besides the well-documented effects on T lymphopoiesis, estrogen also inhibits
cutaneous T cell dependent delayed type hypersensitivity (DTH) reaction [24, 25]. In the skin DTH model, a hapten is applied topically on abdominal skin whereby sensitization occurs. The immunogenic hapten-host protein complex is presented to T cells by antigen-presenting cells (APCs), ultimaltely resulting in clonal T cell proliferation. Reapplied hapten induces the elicitation phase where primed T cells migrate rapidly to the hapten-exposed skin, produce cytokines and induce inflammation [26]. We have previously reported that, in contrast to E2,
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ral cannot inhibit the DTH reaction [24]. The effects of las and bza on T cell development and function needs further investigation, therefore, the aim of this study was to explore the role of
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las and bza on T lymphopoiesis and cutaneous T cell dependent inflammation.
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3. Materials and Methods 3.1 Mice
This study was approved by the ethical committee for animal experiments in Gothenburg. Female C57Bl6N mice (Scanbur, NOVA-SCB AB, Sollentuna, Sweden) were kept in groups
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of 4-5 animals per cage under standard environmental conditions and fed soy-free chow and
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tap water ad libitum.
3.2 Ovariectomy
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Mice were ovariectomized (ovx) at the age of 8 weeks. Surgeries were performed under full anesthesia using Isoflurane (Baxter Healthcare Corporation Chicago, Illinois, USA). Ovaries
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were removed through a midline skin incision followed by flank incisions of the peritoneum, and the skin was closed with metallic clips. Mice received Carprofen (Rimadyl®, Orion
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Pharma Animal Health, Sollentuna, Sweden) as post-operative analgesia and were allowed to
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rest for 10 days after surgery before initiation of treatment.
3.3 Hormones and treatment For the studies of T lymphopoiesis, mice were subcutaneously injected for 16 consecutive days with 17β-estradiol-3-benzoate ; 1 μg/mouse/day, Sigma-Aldrich, St Louis, MO, USA), raloxifene (ral; 60 μg/mouse/day, Sigma-Aldrich), lasofoxifene (las; 4 μg/mouse/day, Pfizer Inc, NY, USA) or bazedoxifene (bza; 24 μg/mouse/day, Pfizer) dissolved in 100 μl miglyol oil (Vendico Chemical AB, Malmö, Sweden). Bza and las were kind gifts from
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Pfizer. Control mice received miglyol oil only (100 μl /mouse/day). It has previously been shown that mice treated with E2 in doses similar to the one used here obtain serum E2 levels corresponding to low diestrus levels (10-25 pg/ml) [27]. The doses of ral las and bza, were
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chosen based on our previous finding that they cause a significant increase in total bone
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mineral density in ovx mice [12]. In addition, body surface area calculations ensured that doses of ral, las and bza used in mice were similar to those used in humans [28].
3.4 DTH reaction
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Treatment with E2, ral, las and bza (doses as described above), was initiated 1 day before sensitization with the hapten oxazolone (OXA; Sigma-Aldrich) and administered daily until the mice were terminated 24 hours after OXA challenge. Mice were sensitized by topical
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application of 150 l of 3% OXA in ethanol:acetone (3:2) on the abdomen. Six days later, the
mice were challenged through cutaneous application of 30 l of 1% OXA in ethanol,
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followed by 30 l of 1% OXA in miglyol oil (Vendico Chemical AB, Malmo, Sweden) on
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both sides of the right ear. Ear thickness was measured using a caliper (Kröplin, Hessen, Germany) 24 hours after challenge and the difference in thickness between the challenged ear
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and the unchallenged ear constituted the DTH reaction. The “no DTH” group was sensitized
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and challenged with the diluents without OXA, and treated with vehicle to serve as a control group.
3.5 Tissue collection and single cell preparation At termination, mice were anesthetized with ketamine (Ketalar®, Pfizer) and medetomidine (Domitor®, Orion Pharma), bled, and killed by cervical dislocation. Thymi were dissected, weighed and mashed through 70m filters. Cells were then washed in PBS, re-suspended in FACS-buffer containing PBS, 10% FCS and 1% sodium azide and counted using an
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automated cell counter (Sysmex Europe GmbH, Nordenstedt, Germany). In the DTH experiment, the challenged ear was dissected, split into a dorsal and a ventral part and digested in 2g/ml Collagenase IV (Sigma-Aldrich) for 1 hour at 37oC. The tissue was
3.6 Flow cytometry analysis
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counted using an automated cell counter (Sysmex).
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mashed through a 70m filter, washed in PBS and resuspended in FACS-buffer and
Thymocytes (0.5×106) were stained with CD4-v450, CD25-APC (BD) and with CD8-FITC, CD44-PerCP (BioLegend), and cells from the ear were stained with CD45-PE, CD11b-v450,
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Ly6C-APC-Cy7, TCR-FITC, CD19-PE, CD8-v500, CD4-v450 (BD) and with CD11c-
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APC, Ly6G-FITC, MHCII-PerCP, CD45-PerCP (BioLegend). Thymocytes were analysed in a FACSCanto II (BD) and cells from the ear in a FACSVerse (BD). All data were further
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processed using Flow Jo version 10.0.4 (Three Star Inc, Ashland, USA). Doublets were excluded using a singlet gate. Results are presented as percentage of the different populations
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per lymphocytes (thymus) or live cells (ear).
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3.7 Statistical analysis
Statistical analyses were performed using IBM SPSS software version 21 (IBM, Armonk,
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NY, USA). Normality was graphically assessed. When positive skewing of the normality was found, the values were logarithmically transformed before analysis. Experiments were terminated on different days; therefore variation between days was assessed and corrected for when needed using ANCOVA, otherwise ANOVA was used. All groups were compared with vehicle using Dunnett’s post hoc test when equal variance was found, and Dunnett’s T3 post hoc test when Levene’s test revealed unequal variance. Data are presented as arithmetic
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mean+SEM when values were linear and as geometric mean+SEM when values were logarithmic. P values < 0.05 were considered statistically significant.
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4. Results 4.1 E2, ral, and las decrease thymus weight
In order to study the effects of E2 and SERMs on thymus, mice were treated for 16
consecutive days with E2, ral, las, bza or vehicle. We have previously shown that the
administrated doses of E2 and SERMs protect against ovx-induced bone loss [12]. At
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termination, the thymus was weighed and the number of thymocytes was determined. In
conjunction with earlier studies [24], E2 and ral both decreased the thymus weight, expressed
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as percentage of total body weight (Fig. 1a). In addition, las significantly reduced the thymus weight, however bza lacked this effect (Fig. 1a). Treatment with E2 decreased the thymic
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cellularity but none of the SERMs altered the total number of thymocytes (Fig. 1b).
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4.2 E2, but not SERMs, alters thymic T cell populations
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To investigate how SERMs influence the composition of thymic T cell populations, thymocytes were characterized by flow cytometry using CD4 and CD8 to obtain the earliest
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CD4-CD8- double negative (DN) population, the subsequent CD4+ CD8+ double positive (DP) population and the most mature CD4+ single positive (SP) and CD8+ SP populations (Fig. 2a). The DN population was divided into DN1-DN4 cells using CD25 and CD44, where DN1 cells were gated as CD25-CD44+, DN2 cells as CD25+CD44+, DN3 cells as CD25+CD44-, and DN4 cells as CD25-CD44- (Fig. 2a). Percentages of these populations after treatment with vehicle, E2 or SERMs are presented in figure 2b-h. Treatment with E2 resulted in an increase in the DN1 population, and a decrease in the DN2 and DN3 populations, while the DN4 population remained unchanged. None of the SERMs altered the DN subpopulations (Fig. 2b-
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e). In addition, treatment with E2 resulted in a tendency towards decreased DP population (Fig. 2f), significantly increased CD4+ SP cells (Fig. 2g), and a tendency towards increased CD8+ SP cells (Fig. 2h). However, the DP and both SP populations were unchanged after
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treatment with SERMs (Fig. 2f-h).
4.3 E2, but not SERMs, suppresses T cell dependent DTH
Mice were subjected to OXA-induced skin DTH and treated with E2, ral, las, bza or vehicle from one day before sensitization until termination 24 hours after challenge. Before
termination, the untreated and hapten-treated ears were measured and the difference in
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thickness was calculated, rendering a DTH reaction in mm 10-2. In accordance with earlier
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studies [24], treatment with E2 mediated suppression in the DTH reaction against OXA, while this effect was not seen with ral. Similarly to ral, treatment with las or bza did not suppress
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DTH (Fig. 3).
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4.4 Effects of E2 and SERMs on leukocyte populations in the ear after challenge with OXA
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In order to study if any of the treatments altered the composition of immune cells in the ear after induction of DTH, cells from the ear challenged with OXA were subjected to flow
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cytometry. Cells were gated as CD45+ to include leukocytes only and further divided into CD3+ and CD19+ cells to analyze T and B cells, respectively. The total leukocyte population, as well as T and B cells, were increased in the vehicle-treated DTH group compared with the vehicle-treated control group that was not subjected to DTH (Fig. 4b-d). However, no changes in the frequencies of total leukocytes, B or T cells in the challenged ear were seen with either E2 or SERM treatment (Fig. 4b-d). In addition, innate immune cells were studied by further dividing CD45+ cells into neutrophils (CD11b+Ly6G+LY6C-), macrophages (CD11b+Ly6G-
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Ly6C+), CD11c+ cells and T cells (TCR+). Neutrophils, macrophages, CD11c+ cells, and
T cells were significantly enriched in the vehicle-treated DTH group compared with the
vehicle-treated control group that was not subjected to DTH. The CD11c+ cell population was
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decreased after treatment with E2 and ral, but no other differences were found with any of the
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treatments (Fig. 5b-e).
5. Discussion
In the present study we have investigated the effects of the third generation SERMs las and
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bza on T cell development in thymus and on hapten-induced DTH – a T cell dependent skin inflammation. We demonstrate that las, similarly to the second generation SERM ral,
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mediates a minor thymic atrophy but that bza completely lacks effects on thymus weight and cellularity. Furthermore, we show that in contrast to E2, neither las nor bza alter thymic T cell
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populations, or suppress inflammation in DTH.
Thymic T cell development occurs in a stepwise manner and requires dynamic
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migration of T cell progenitors within the thymus and signals from stromal cells, such as cortical epithelial cells [29]. E2 has previously been shown to increase the DN1 cells and
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decrease the DN2 cells implying a block in T cell development at the earliest stage [7].
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Furthermore, E2 decreases the frequency of DP cells while it increases the frequencies of the most mature CD4+ and CD8+ SP cells [7, 24]. In addition, E2 treatment decreases the thymic cortex area and reduces epithelial cells in the thymic cortex [24, 30]. The second generation SERM ral does not diminish the cortex area and only gives a slight decrease in cortical epithelial cells [24]. Furthermore, ral does not alter the thymic T cell populations [24]. In line with previous studies, we confirmed that E2 caused an increase in the DN1 population and a decrease in the following DN2 and DN3 populations, implying a block in the transition of DN1 to DN2/DN3 developmental stages. Moreover, the proportion of CD4+ SP cells was
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increased by E2, but opposite to previous studies, we did not find a significant decrease in the DP population or an increase in the CD8+ SP population. The discrepancy between our results and previous studies is most likely due to the lower, more physiological, dose of E2 used in
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our study.
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None of the SERMs used in this study influenced populations of developing T cells in the thymus. Thymic T cell development is promoted by signals from cortical epithelial cells, and previous studies have shown effects of E2, but not ral, on these cells [24, 30]. Therefore, it is reasonable to speculate that also las and bza have limited effects on cortical epithelial
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cells and that the divergent effects of SERM and E2 on cortical epithelial cells might explain the differences in the compounds’ abilities to influence T cell development. However, this
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remains to be investigated. Treatment with E2 also reduces the number of thymus-homing lymphoid progenitors in the bone marrow leading to decreased influx of early T cell
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progenitors to the thymus [21]. Whether the SERMs affect these cells in the bone marrow or migration of lymphoid progenitor cells to the thymus is the subject of further investigation.
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Studies using knock-out mice lacking the different estrogen receptors have established that estrogen receptor alpha (ER) is indispensable for E2-mediated alterations of thymic T
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cell populations but that ERonly partly mediates E2-induced thymic atrophy [20]. For full
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thymus involution, both estrogen receptor beta (ER) and the transmembrane-bound estrogen receptor GPR30 are required, in addition to ER[20, 31, 32]. Las and bza bind with high affinity to both ERα and ERβ [33, 34], but it is unclear whether they bind to GPR30. The DTH reaction is characterized by a sensitization phase involving antigenpresentation to naïve T cells, and an elicitation phase where the primed T cells migrate to the place of hapten application and induce inflammation [35, 36]. The mechanism for the suppressive effects of E2 on DTH is not clear, but E2 is not believed to directly affect T cells in DTH as female severe combined immunodeficient (SCID) mice reconstituted with
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thymocytes from E2-treated mice did not show a decrease in the DTH response [37]. Instead, the E2-mediated inhibition of DTH is believed to involve a decrease in the antigen presentation to T cells and an alteration of the cytokine pattern [38, 39]. Similarly, E2
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treatment reduces the ability of splenic DCs to present antigen to T cells and to produce
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proinflammatory cytokines in other inflammatory models [40]. Possibly, SERMs lack the
suppressive effects on antigen presentation, which could explain the discrepancy between E2 and SERMs in effects on DTH.
Estrogen plays a complex role in autoimmunity and the prevalence of many
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autoimmune diseases has a clear female preponderance. E2 treatment increases the disease progression in systemic lupus erythematosus [41-43]. Conversely, hormone replacement
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therapy including E2 ameliorates rheumatoid arthritis and multiple sclerosis [44, 45] and treatment with E2 inhibits experimental models of arthritis and multiple sclerosis [8-10]. Our
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group has previously demonstrated that ral ameliorates arthritis and protects against bone loss in experimental collagen-induced arthritis (CIA) [11]. Interestingly, preliminary data show
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that also las and bza have beneficial effects on arthritis and associated bone loss in CIA (Andersson et al, submitted). T and B cells are both important in the pathogenesis of CIA.
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Since las and bza lack effects on T cell dependent inflammation in the DTH model but ameliorates CIA, anti-arthritic effects might be attributed to SERM-mediated modulation of B
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cells or antigen presenting cells instead. In conclusion, this study characterized the effects of the third generation SERMs las
and bza on T cells and reveals that las and bza completely lack effects on T lymphopoiesis, and T cell dependent inflammation in a DTH model. Characterization of immunomodulatory effects of the anti-osteoporotic SERMs is important in several aspects: subclinical inflammation is a component in postmenopausal osteoporosis, and SERMs might be beneficial in treatment of arthritis and associated inflammation-induced bone loss.
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6. Acknowledgements We thank Merja Nurkkala-Karlsson, Maria Bergquist, Sofia Andersson and Malin Erlandsson
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for excellent laboratory assistance and Christina Björklund for animal care.
Author contributions:
AB had full access to all of the data in the study and take responsibility for the integrity and accuracy of the data. The authors are justifiably credited with authorship, according to the
Study design. AB, AA, HC, UI
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Acquisition of data. AB, AA, LG, AS, UI,
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authorship criteria. In detail:
Analysis and interpretation of data. AB, AA, LG, HC, UI
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Statistical analysis. AB, AA, LG, UI
Writing of manuscript. AB, AA, LG, AS, HC, UI
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All authors have given their final approval of the manuscript.
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Funding
This study was supported by grants from The Medical Faculty of the University of
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Gothenburg, the Gothenburg Medical Society, COMBINE, the Swedish Research Council, King Gustav V’s 80 years’ foundation, the Association against Rheumatism, the Swedish Association for Medical Research and LUA/ALF, the Wilhelm and Martina Lundgren Science Foundation 1, the Lars Hierta Foundation, the Magnus Bergvall Foundation, the family Thöléns and Kristlers Foundation, the Åke Wiberg Foundation. The FACS Canto II and the FACS Verse instrument have been bought thanks to generous support from the IngaBritt and Arne Lundberg Foundation.
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7. Conflict of Interest
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The authors declare no conflict of interest.
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Figure legends Figure 1. E2, ral, and las but not bza reduce thymus weight Thymus weight expressed as percentage of total body weight (a) and thymus cellularity (b)
us cr ip
t
after 16 days of treatment, n = 8-10 mice/group. Data are expressed as mean + SEM. (** p < 0.01, *** p < 0.001).
Figure 2. E2 but not SERMs alters thymic T cell populations
(a) Gating strategy for DN1-4, DP and SP cells in thymus. Percentage of thymic lymphocytes
an
of DN1 (b), DN2 (c), DN3 (d), DN4 (e), DP (f), CD4+ SP (g), CD8+ SP (h) in thymus after 16 days of treatment, n = 8-10 mice/group. Data are expressed as mean + SEM (* p < 0.05, ** p
M
< 0.01).
ed
Figure 3. E2 but not SERMs suppress DTH
Difference in mm 10-2 in ear thickness between ear treated with OXA and untreated ear in
pt
mice subjected to DTH and treated with E2 or SERMs, n = 8-10 mice/group. Data are
ce
expressed as mean + SEM (** p < 0.01, *** p < 0.001).
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Figure 4. Treatment with E2 or SERMs does not alter the percentage of adaptive immune cells in ear after DTH induction OVX mice were treated with E2 or SERMs and subjected to DTH. Cells from inflamed ears were analysed by flow cytometry. (a) Gating strategy for ear skin T and B cells: a small live gate was made and CD45+ cells were further divided into CD19+ cells (B cells) and CD19cells. The CD19- population was then used to gated on CD3+ cells (T cells). Percentage of CD45+ cells (b), CD19+ cells (c) and CD3+ cells (d) of live singlet cells, n = 7-10 mice/group. Data are expressed as mean + SEM (* p < 0.05, ** p < 0.01). 17
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Figure 5. E2 and ral reduce CD11c+ cells in ear after DTH induction
t
OVX mice were treated with E2 or SERMs and subjected to DTH. Cells from inflamed ears
us cr ip
were analysed by flow cytometry. (a) Gating strategy for ear skin innate immune cells: a live cell gate was made and CD45+ cells were further divided into TCR+ cells (T cells),
CD11c+ cells and CD11b+ cells. CD11b+ cells were further divided into Ly6G+Ly6C+ cells (neutrophils) and Ly6G-Ly6C+ cells (macrophages). Percentage of CD45+ cells (b),
an
macrophages and monocytes (c), neutrophils (d), CD11c+ cells (e) and T cells (f) of live singlet cells, n = 7-10 mice/group. Data are expressed as mean + SEM (* p < 0.05, ** p
