Journal of Reproductive Immunology 106 (2014) 58–66
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Hormonal regulation of the cytokine microenvironment in the mammary gland Pallave Dasari a,b,c,1 , David J. Sharkey b,c,1 , Effarina Noordin b,c , Danielle J. Glynn a,b,c , Leigh J. Hodson a,b,c , Peck Y. Chin b,c , Andreas Evdokiou a , Sarah A. Robertson b,c , Wendy V. Ingman a,b,c,∗ a b c
Discipline of Surgery, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Woodville, Australia School of Paediatrics and Reproductive Health, University of Adelaide, Australia Research Centre for Reproductive Health, The Robinson Institute, University of Adelaide, Australia
a r t i c l e
i n f o
Article history: Received 23 April 2014 Received in revised form 4 July 2014 Accepted 5 July 2014 Keywords: Mammary gland Oestrogen Progesterone Cytokines Inflammation
a b s t r a c t The mammary gland is a unique organ that undergoes hormone-driven developmental changes over the course of the ovarian cycle during adult life. Macrophages play a role in regulating cellular turnover in the mammary gland and may affect cancer susceptibility. However, the immune microenvironment that regulates macrophage function has not been described. Hormonal regulation of the cytokine microenvironment across the ovarian cycle was explored using microbead multiplex assay for 15 cytokines in mammary glands from C57Bl/6 mice at different stages of the oestrous cycle, and in ovariectomised mice administered oestradiol and progesterone. The cytokines that were found to fluctuate over the course of the oestrous cycle were colony-stimulating factor (CSF)1, CSF2, interferon gamma (IFNG) and tumour necrosis factor alpha (TNFA), all of which were significantly elevated at oestrus compared with other phases. The concentration of serum progesterone during the oestrus phase negatively correlated with the abundance of cytokines CSF3, IL12p40, IFNG and leukaemia inhibitory factor (LIF). In ovariectomised mice, exogenous oestradiol administration increased mammary gland CSF1, CSF2, IFNG and LIF, compared with ovariectomised control mice. Progesterone administration together with oestradiol resulted in reduced CSF1, CSF3 and IFNG compared with oestradiol administration alone. This study suggests that the cytokine microenvironment in the mammary gland at the oestrus phase of the ovarian cycle is relatively pro-inflammatory compared with other stages of the cycle, and that the oestradiol-induced cytokine microenvironment is significantly attenuated by progesterone. A continuously fluctuating cytokine microenvironment in the mammary gland presumably regulates the phenotypes of resident leukocytes and may affect mammary gland cancer susceptibility. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction ∗ Corresponding author at: Discipline of Surgery, The Queen Elizabeth Hospital DX465702, 28 Woodville Road, Woodville 5011, Australia. Tel.: +61 8 8222 6141; fax: +61 8 8222 6076. E-mail address: [email protected] (W.V. Ingman). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jri.2014.07.002 0165-0378/© 2014 Elsevier Ireland Ltd. All rights reserved.
During the ovarian cycle, the mammary gland epithelium undergoes proliferation, differentiation and apoptosis under the direction of ovarian hormones oestrogen and progesterone (Fata et al., 2001; Ramakrishnan et al., 2002), and this cellular turnover has been linked to the high
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susceptibility of the mammary gland to tumour formation (Ramakrishnan et al., 2002). During the oestrus phase of the cycle, the mammary epithelium exhibits a basic ductal structure. As serum progesterone rises, secondary branching and alveolar bud development occur, with alveolar bud appearance predominantly during dioestrus (Fata et al., 2001). If pregnancy does not proceed, the fall in progesterone at proestrus induces apoptosis of the newly formed alveolar buds, and the tissue is remodelled back to the basic architecture. Macrophages are immune system cells with multiple roles in epithelial cell turnover during the course of the ovarian cycle (Chua et al., 2010). Macrophages promote oestradiol- and progesterone-induced epithelial cell proliferation and alveolar development following ovulation, and when progesterone levels fall towards the end of the cycle, macrophages promote phagocytosis of apoptotic cells and remodel the tissue to its rudimentary structure. These differing macrophage functions are associated with altered phenotype, with fluctuation in expression of MHCII, CD204 and NKG2D by mammary gland macrophages across the cycle under hormonal control (Hodson et al., 2013). In order to recruit macrophages and regulate specific phenotypes and functions of these cells, an array of cytokines and chemokines are likely to be present in the mammary gland microenvironment. Cytokines comprise the interleukins, lymphokines and related regulatory glycoproteins released by cells of the immune system or non-haematopoietic cells, to act as intercellular mediators in the generation of an immune response. Previous studies have shown that cytokines, including colony-stimulating factor 1 (CSF1), interleukin 4 (IL4) and interleukin 13 (IL13), play important roles in mammary gland development during puberty and pregnancy (Pollard and Hennighausen, 1994; GouonEvans et al., 2000; Ingman et al., 2006; Khaled et al., 2007). CSF1, for example, is known to be a regulator of macrophage survival. Csf1 null mutant mice have significantly reduced macrophage abundance and display impaired mammary gland development during puberty and lactation (Gouon-Evans et al., 2000; Ingman et al., 2006). IL4 and IL13 play a role in the promotion of luminal mammary epithelial cell development during pregnancy through the Stat6/Gata3 signalling pathway. Mice with a double mutation in both the Il4 and Il13 genes show a significant decrease in the number of mammary gland side branches and alveolar buds (Khaled et al., 2007). Increased breast cancer risk is associated with early menarche, late menopause and increased cumulative number of menstrual cycles (Chavez-Macgregor et al., 2008; Collaborative Group on Hormonal Factors in Breast Cancer, 2012). Circulating oestrogens are elevated in young women with early menarche (Shi et al., 2010) and this early exposure to oestrogen may be responsible for the increased risk of breast cancer in women with early menarche over and above the increased risk associated with menstrual cycling (Collaborative Group on Hormonal Factors in Breast Cancer, 2012). Fluctuating ovarian hormones presumably create a pro-tumourigenic environment in the
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mammary gland that, over time, increases the risk of cancer. The biological factors behind this pro-tumourigenic microenvironment have yet to be elucidated, but may include impaired immune surveillance and/or inflammation associated with altered cytokine regulation (Need et al., 2014). The aim of this study was to define the cytokine microenvironment across the four phases of the mouse ovarian cycle and to investigate hormonal regulation of cytokines in the mammary gland. We demonstrate that the concentration of specific cytokines in the mammary gland fluctuates over the course of the ovarian cycle, with the oestrus phase being associated with a relatively pro-inflammatory cytokine microenvironment compared with other stages of the cycle. Furthermore, circulating progesterone negatively correlates with a number of these cytokines at the oestrus phase of the cycle, and in ovariectomised mice progesterone suppresses the oestradiol-induced cytokine profile. Fluctuations in the cytokine microenvironment in the mammary gland over the course of the ovarian cycle may affect susceptibility to mammary gland tumourigenesis. 2. Materials and methods 2.1. Mice All animal experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition, 2004). Mice were maintained in specific pathogen-free conditions with controlled light (12 h light, 12 h dark cycle) and temperature at the Laboratory Animal Services Medical School facility. The oestrous stages of naturally cycling C57Bl/6 mice were determined using cytological characteristics of vaginal smears (Bronson et al., 1968) performed daily. The mice were tracked for a minimum of two weeks through at least one normal four- to five-day oestrous cycle and were 12–15 weeks old at mammary gland dissection. Mice were euthanised at each of the four stages of the oestrous cycle (oestrus, n = 16; metoestrus, n = 20; dioestrus, n = 18; and proestrus, n = 14). At the time of euthanasia, blood was collected by cardiac puncture and the fourth abdominal pair of mammary glands was dissected, snap frozen in liquid nitrogen and stored at −80 ◦ C before protein extraction. To investigate hormonal regulation of the mammary gland cytokine microenvironment, 12-week-old mice were ovariectomised under 2% isoflurane anaesthesia and rested for one week. Subcutaneous 17-oestradiol (1 g) alone or in combination with progesterone (1 mg) was administered in 100 l of sesame oil daily for three days, while control mice were given vehicle alone. On the fourth day, approximately 1 mL of blood was collected by cardiac puncture and the fourth pair of inguinal mammary glands was dissected. The section between the lymph node and the distal end of the gland was snap frozen in liquid nitrogen and stored at −80 ◦ C for cytokine analysis. Blood was centrifuged for six minutes at 350 g and serum was collected and stored at −80 ◦ C.
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2.2. Cytokine analysis Protein was extracted from the mammary gland tissue, using EDTA-free protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA) dissolved in 10 mL of protein extraction buffer containing 500 mM Tris–HCl (Sigma–Aldrich, Castle Hill, NSW, Australia), 200 mM NaCl (Merck, Darmstadt, Germany) and 10 mM CaCl2 (BDH Chemicals, Murarrie, QLD, Australia). The mammary gland was homogenised in 1 mL of the extraction buffer, centrifuged and the aqueous layer collected. After adding 10% Triton X-100 (Bio-Rad Laboratories, Hercules, CA, USA) to the aqueous layer, the solution was homogenised again, the aqueous layer collected and stored at −80 ◦ C. Protein concentration of the sample was measured using Nanodrop ND-100 (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The concentration of cytokines in mammary gland extracts was quantified using Luminex xMAP® technology (Millipore, Billerica, MA, USA), a bead-based multiplex platform for the detection of multiple proteins in a single sample, as per the manufacturer’s instructions. The following cytokines were selected: colony-stimulating factor-1 (CSF1), CSF2, CSF3, interleukin (IL) 1 beta (IL1B), IL2, IL4, IL5, IL6, IL10, IL12p40, IL13, IL17, interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and leukaemia inhibitory factor (LIF). All samples were measured in a single assay. The intra-assay coefficients of variation were less than 10%. The lower detection limits of the assays were 1.1 pg/mL CSF1, 5.6 pg/mL CSF2, 0.9 pg/mL CSF3, 3.2 pg/mL IL1B, 3.2 pg/mL IL2, 0.4 pg/mL IL4, 1.8 pg/mL IL5, 3.3 pg/mL IL6, 3.2 pg/mL IL10, 4.9 pg/mL IL12p40, 3.2 pg/mL IL13, 0.5 pg/mL IL17, 0.9 pg/mL IFNG, 1.0 pg/mL TNFA and 0.8 pg/mL LIF. The concentrations of cytokines measured are presented as picograms per milligramme of protein (pg/mg).
2.3. Serum hormone analysis Oestradiol and progesterone concentrations in serum were measured using commercial RIA kits DSL-4800 and DSL-3400 respectively (Diagnostic Systems Laboratories, Webster, TX, USA) according to the instructions of the manufacturer. All samples were measured in a single assay. The within-assay coefficients of variation were 10.0% in the oestradiol assay and 7.9% in the progesterone assay. The lower limits of detection were 5 pg/mL of oestradiol and 0.3 ng/mL of progesterone.
2.4. Statistical analysis Data were analysed using SPSS Statistics version 20 (IBM Software, New York, NY, USA). The non-parametric Kruskal–Wallis test was conducted, followed by post hoc analysis with Mann–Whitney U test for comparisons of cytokine concentrations among multiple groups. Spearman’s rho test was employed to examine correlations between progesterone and specific cytokines during oestrous phases. Significance was inferred at p < 0.05.
Table 1 Oestradiol and progesterone concentrations in serum during phases of the oestrous cycle in naturally cycling C57/Bl6 mice. Values are mean ± standard error, n = 14–20 per group. Statistics analysis using Kruskal–Wallis test with post hoc Mann–Whitney U test, p < 0.05. Groups assigned ‘a’ differ significantly from groups assigned ‘b’ and ‘c’. Estrous phase
Estradiol (pg/mL)
Oestrus Metoestrus Dioestrus Proestrus
41.3 31.5 34.3 27.3
± ± ± ±
3.3a 2.8ab 3.0ab 1.7b
Progesterone (ng/mL) 1.5 5.9 4.8 2.4
± ± ± ±
0.1a 1.0b 1.2bc 0.5ac
3. Results 3.1. Serum hormone concentration in naturally cycling mice Levels of serum oestradiol and progesterone varied across the phases of the oestrous cycle, as expected (Table 1). Oestradiol peaked during oestrus and was significantly elevated compared with proestrus. Serum progesterone was lowest at the oestrus phase and was significantly increased during metoestrus and dioestrus. 3.2. Fluctuation in cytokine concentration in the mammary gland across the oestrous cycle Cytokine analysis by microbead technology was used to determine the concentration of 15 cytokines in the mammary gland during the oestrous cycle. Of these, IL5, IL6 and IL17 were not detected in the mammary gland at any time during the oestrous cycle (data not shown). The remaining 12 cytokines (CSF1, CSF2, CSF3, IL1B, IL2, IL4, IL10, IL12p40, IL13, IFNG, TNFA and LIF) were all present at detectable levels in mammary gland protein extracts. Since several cytokines showed evidence of fluctuations over the course of the oestrous cycle, the relationship between serum oestradiol and progesterone with mammary gland cytokine concentration was analysed. For all 12 detectable cytokines, no significant correlations with serum oestradiol were detected during any phase (data not shown). During the dioestrus and proestrus phases of the cycle, there were no correlations between cytokines and progesterone (data not shown). However, negative correlations between progesterone and a number of different cytokines were observed during oestrus and metoestrus. The correlations between progesterone and cytokines during the oestrus phase of the cycle were most striking, and are presented for each cytokine in Figs. 1–3. The significant correlations observed at metoestrus are presented in the text below. Concentrations of CSF1 and CSF2 were significantly elevated at oestrus compared with metoestrus, dioestrus and proestrus (Fig. 1A and B respectively). CSF3 concentration did not significantly fluctuate across the oestrous cycle (Fig. 1C). At oestrus, CSF1 and CSF2 had no significant relationship with progesterone (Fig. 1A and B); however, CSF3 negatively correlated with progesterone (rs = −0.55; p = 0.028; Fig. 1C). The concentration of CSF2 showed a negative correlation with progesterone during the metoestrus phase of the cycle (rs = −0.48; p = 0.031).
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P 4 ( n g /m l) Fig. 1. Colony-stimulating factor (CSF) concentrations in mammary gland of mice. Mammary glands from naturally cycling C57/Bl6 mice were harvested at different phases of the oestrous cycle and protein extracted to measure with Luminex xMAP Technology (A) CSF1, (B) CSF2 and (C) CSF3, as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) CSF1, (B) CSF2 and (C) CSF3, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
Six interleukins (IL1B, IL2, IL4, IL4, IL12p40 and IL13) were detected continuously over the course of the oestrous cycle, with no significant variation between phases (Fig. 2). However, mammary gland IL-12p40 concentration exhibited a significant negative correlation with progesterone during the oestrus phase of the cycle (rs = −0.54; p = 0.033).
At metoestrus, progesterone negatively correlated with IL2 (rs = −0.65; p = 0.002), IL4 (rs = −0.51; p = 0.022), IL10 (rs = −0.50; p = 0.025), IL12p40 (rs = −0.47; p = 0.038). The concentration of IFNG was significantly increased at oestrus compared with metoestrus, dioestrus and proestrus (Fig. 3A). TNFA concentration was significantly
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Fig. 2. Interleukin (IL) concentrations in the mammary glands of mice. Interleukin concentrations measured in mammary glands from naturally cycling C57/Bl6 mice were harvested during different phases of the oestrous cycle; (A) IL1B, (B) IL2, (C) IL4, (D) IL10, (E) IL12p40 and (F) IL13, as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) IL1B, (B) IL2, (C) IL4, (D) IL10, (E) IL12p40 and (F) IL13, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
higher during oestrus compared with dioestrus and proestrus (Fig. 3B). LIF concentration did not significantly fluctuate across the cycle (Fig. 3C). IFNG and LIF concentrations show negative correlation with progesterone during the oestrus phase of the cycle (rs = −0.56; p = 0.025 and rs = −0.57; p = 0.023 respectively). 3.3. Hormonal regulation of mammary gland cytokines Circulating progesterone appeared to have a profound negative regulatory effect on mammary gland cytokine concentration in naturally cycling mice. This was particularly evident during the oestrus phase of the cycle when circulating oestradiol peaks, and the concentration of a number of cytokines are increased. This suggests that oestradiol induces production of these cytokines, while progesterone dampens this effect. To investigate the effect of ovarian hormones on mammary gland cytokine concentration, ovariectomised mice were treated with vehicle control, oestradiol only, or oestradiol together with progesterone. Further microbead analysis of cytokines was
conducted on those cytokines shown to fluctuate across the natural cycle or to be negatively regulated by progesterone during the oestrus phase. Mammary gland CSF1 concentration in oestradioltreated mice was significantly higher than in control mice and in those treated with oestradiol and progesterone (Fig. 4A). In contrast, CSF2 concentration was significantly lower in mammary glands from oestradiol-treated mice than in those from mice treated with oestradiol and progesterone (Fig. 4B). CSF3 concentration was decreased in mammary glands from mice treated with oestradiol and progesterone compared with oestradiol-treated mice (Fig. 4C). The concentration of IFNG in mammary glands from oestradiol-treated mice was significantly elevated compared with those from control mice and mice treated with oestradiol and progesterone (Fig. 4E). Concentration of LIF was higher in oestradiol-treated mice than in control mice (Fig. 4G). There was no significant effect of oestradiol or progesterone on IL12p40 (Fig. 4D) or TNFA (Fig. 4F) concentration in mammary gland protein extracts.
P. Dasari et al. / Journal of Reproductive Immunology 106 (2014) 58–66
A. 0 .5
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P 4 ( n g /m l) Fig. 3. Proinflammatory cytokine concentrations in the mammary glands of mice. Cytokine concentrations measured in mammary glands from naturally cycling C57/Bl6 mice were harvested during different phases of the oestrous cycle: (A) interferon gamma (IFNG), (B) tumour necrosis factor (TNFA) and (C) leukaemia inhibitory factor (LIF), as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) IFNG, (B) TNFA and (C) LIF, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
4. Discussion A number of cytokines have been ascribed important roles in the events of mammary gland development and remodelling during puberty, pregnancy, lactation and involution (Pollard and Hennighausen, 1994; Gouon-Evans et al., 2000; Ingman et al., 2006; Khaled et al., 2007; Ingman
and Robertson, 2008; Watson, 2009; O’Brien et al., 2010). However, the abundance and types of cytokines in the adult cycling mammary gland have not been investigated. In part this is because the gland is relatively quiescent in non-pregnant adult females, compared with the extensive morphogenesis that occurs during puberty, pregnancy and involution. However, there are indeed structural and
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Fig. 4. Cytokine production in response to hormonal regulation. C57/Bl6 mice were ovariectomised and treated with control, oestradiol (E2) or oestradiol and progesterone (E2 + P4) for three days before mammary glands were harvested and measured (A) CSF1, (B) CSF2, (C) CSF3, (D) IL12p40, (E) IFNG, (F) TNFA and (G) LIF. Bars represent mean values, n = 7–12 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups (*p < 0.05).
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immunological changes that accompany each phase of the oestrous cycle. In particular, previous studies in mice have demonstrated that macrophage phenotype and function fluctuate across the ovarian cycle, and are regulated by the ovarian hormones oestradiol and progesterone (Chua et al., 2010; Hodson et al., 2013), suggesting that there might be accompanying changes in the cytokine microenvironment. In this study we provide evidence of fluctuating cytokine activity in the mammary gland microenvironment throughout the ovarian cycle. IFNG, TNFA, CSF1 and CSF2 concentrations were elevated during oestrus when circulating oestradiol peaked, and declined during metoestrus, dioestrus and proestrus. Although exogenous oestradiol administration caused elevation in a number of cytokines in ovariectomised mice, cytokine abundance did not correlate with circulating oestradiol in naturally cycling mice. We observed substantial variation in cytokine abundance during oestrus between mice, and a number of mice analysed at this stage of the cycle did not exhibit elevated cytokine concentration. Further analysis of this variation at oestrus revealed significant negative correlations between circulating progesterone and mammary gland cytokine concentration of CSF3, IL12, IFNG and LIF during this phase of the cycle. Moreover, administration of exogenous progesterone attenuated oestradiol-induced elevation in cytokine abundance in ovariectomised mice. This suggests that although oestradiol induces cytokine production, progesterone has a strong suppressive effect on these specific cytokines, even at the low circulating level of progesterone observed during the oestrus phase of the cycle. Therefore, we propose that the fluctuation in cytokine concentration in the mammary gland across the cycle is due to induction of cytokine production by oestradiol during the oestrus phase of the cycle, which is suppressed by progesterone during the other phases. Results from previous studies suggest that cytokine activity in the mammary gland tends to be associated with active periods of macrophage-assisted morphogenesis. CSF1 is a chemoattractant for macrophages (Van Nguyen and Pollard, 2002) and promotes mammary morphogenesis during puberty, pregnancy and involution (Pollard and Hennighausen, 1994; Gouon-Evans et al., 2000; Ingman et al., 2006; O’Brien et al., 2011). CSF2 is also important in regulating macrophage function, proliferation and survival (Lehtonen et al., 2002). IFNG and TNFA expression in the mammary gland is elevated during pregnancy and decreased during lactation (Varela and Ip, 1996; Khaled et al., 2007). LIF mRNA has been shown to increase in the mammary gland during involution, and may be necessary for regression during early involution by initiating epithelial cell apoptosis (Clarkson et al., 2004). As dioestrus is the most active phase of the cycle in respect of epithelial cell activity (Fata et al., 2001) and macrophage abundance (Chua et al., 2010), with the oestrus phase considered relatively inactive, the elevation in cytokines during the oestrus phase of the cycle and by exogenous oestradiol administration was surprising. The roles of these cytokines in mammary gland development and function at the oestrus phase are unclear; however, our results indicate a highly active immune microenvironment at this time. One possibility is that the elevated cytokines
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associated with oestrus initiate pathways of immune adaptation, which in the event of conception progress to the further morphogenesis and functional changes seen in pregnancy. We have not investigated the cell types in the mammary gland responsible for cytokine synthesis; however, epithelial cells, fibroblasts, adipocytes and immune cells, including macrophages, are all capable of oestradiolinduced cytokine secretion, and may contribute to this cytokine microenvironment. Our results suggest that the pro-inflammatory cytokines TNFA, IFNG and LIF may have under-appreciated roles in the mammary gland. Further studies are required to understand how these cytokines contribute to mammary gland morphogenesis and homeostasis. In many tissues, elevated pro-inflammatory cytokine concentrations are linked to tumour promotion and progression (Karin and Greten, 2005). Pro-inflammatory cytokines, including TNFA and CSF1, can stimulate macrophages to produce nitric oxide and reactive oxygen species, causing DNA damage and mutations. Activation of the NF-kB signalling pathway by TNFA causes elevation in the abundance of pro-inflammatory cytokines, such as IFNG, leading to tumour promotion and progression (Karin and Greten, 2005; Lin and Karin, 2007; Rivas et al., 2008). TNFA, in synergy with oestradiol, promotes the proliferation of breast cancer cells (Rubio et al., 2006) and drug-resistant breast cancer tumours (Pradhan et al., 2010). CSF1 also contributes to excessive inflammatory responses by regulating macrophage function to produce more proinflammatory cytokines (Irvine et al., 2006). Therefore, the increased abundance of these pro-inflammatory cytokines during oestrus may promote a tumourigenic environment. On the other hand, progesterone-regulated immune cells may also have an impact on breast cancer risk. Carcinogenic effects of progesterone are mediated locally within the mammary gland epithelium by the tumour necrosis factor-related cytokine RANK ligand (GonzalezSuarez et al., 2010; Schramek et al., 2010). Production of RANK ligand by infiltrating regulatory T cells promotes mammary cancer progression (Tan et al., 2011), and inhibition of this pathway might be utilised to prevent breast cancer (Gonzalez-Suarez et al., 2010). Progesterone withdrawal appears to inhibit tumour immune surveillance (Hodson et al., 2013), which might be associated with the increased risk of mammary cancer observed in rats injected with chemical carcinogen during the proestrus phase of the ovarian cycle, compared with the dioestrus phase (Ratko and Beattie, 1985). Thus, it seems that both extremes of the hormone fluctuations associated with the ovarian cycle raise the risk of tumour induction and progression through inflammation driving tumourigenesis and through altered immune surveillance. 5. Conclusion Here we report that the cytokine microenvironment in the mammary gland fluctuates over the course of the ovarian cycle and is hormonally regulated. A number of pro-inflammatory cytokines are elevated during the oestrus phase of the cycle. Administration of exogenous
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oestradiol increases pro-inflammatory cytokine concentration, an effect that is largely suppressed by progesterone. These cytokines are likely to directly and/or indirectly regulate macrophage function, phenotype and abundance in the mammary gland throughout the cycle. Further studies are required to dissect the role of these cytokines in mammary gland morphogenesis, homeostasis and breast cancer risk. Funding This work was supported by National Breast Cancer Foundation, THRF Postdoctoral Fellowship (PD), MichellMcGrath THRF Fellowship (AE) and NBCF/THRF Fellowship (WVI). Conflict of interest statement None declared. References Bronson, F.H., Dagg, C.P., Snell, G.D., 1968. Reproduction. In: Green, E.L. (Ed.), Biology of the Laboratory Mouse. , 2nd ed. Dover Publications, New York. Collaborative Group on Hormonal Factors in Breast Cancer, 2012. Menarche, menopause, and breast cancer risk: individual participant meta-analysis, including 118 964 women with breast cancer from 117 epidemiological studies. Lancet Oncol. 13, 1141–1151. Chavez-Macgregor, M., Van Gils, C.H., Van Der Schouw, Y.T., Monninkhof, E., Van Noord, P.A., Peeters, P.H., 2008. Lifetime cumulative number of menstrual cycles and serum sex hormone levels in postmenopausal women. Breast Cancer Res. Treat. 108, 101–112. Chua, A.C., Hodson, L.J., Moldenhauer, L.M., Robertson, S.A., Ingman, W.V., 2010. Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium. Development 137, 4229–4238. Clarkson, R.W., Wayland, M.T., Lee, J., Freeman, T., Watson, C.J., 2004. Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression. Breast Cancer Res. 6, R92–R109. Fata, J.E., Chaudhary, V., Khokha, R., 2001. Cellular turnover in the mammary gland is correlated with systemic levels of progesterone and not 17beta-estradiol during the estrous cycle. Biol. Reprod. 65, 680–688. Gonzalez-Suarez, E., Jacob, A.P., Jones, J., Miller, R., Roudier-Meyer, M.P., Erwert, R., et al., 2010. Rank ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468, 103–107. Gouon-Evans, V., Rothenberg, M.E., Pollard, J.W., 2000. Postnatal mammary gland development requires macrophages and eosinophils. Development 127, 2269–2282. Hodson, L.J., Chua, A.C., Evdokiou, A., Robertson, S.A., Ingman, W.V., 2013. Macrophage phenotype in the mammary gland fluctuates over the course of the estrous cycle and is regulated by ovarian steroid hormones. Biol. Reprod. 89, 65. Ingman, W.V., Robertson, S.A., 2008. Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects. Biol. Reprod. 79, 711–717. Ingman, W.V., Wyckoff, J., Gouon-Evans, V., Condeelis, J., Pollard, J.W., 2006. Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland. Dev. Dyn. 235, 3222–3229.
Irvine, K.M., Burns, C.J., Wilks, A.F., Su, S., Hume, D.A., Sweet, M.J., 2006. A csf-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations. FASEB J. 20, 1921–1923. Karin, M., Greten, F.R., 2005. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat. Rev. Immunol. 5, 749–759. Khaled, W.T., Read, E.K., Nicholson, S.E., Baxter, F.O., Brennan, A.J., Came, P.J., et al., 2007. The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development. Development 134, 2739–2750. Lehtonen, A., Matikainen, S., Miettinen, M., Julkunen, I., 2002. Granulocyte-macrophage colony-stimulating factor (GM-CSF)induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation. J. Leukoc. Biol. 71, 511–519. Lin, W.W., Karin, M., 2007. A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Invest. 117, 1175–1183. Need, E.F., Atashgaran, V., Ingman, W.V., Dasari, P., 2014. Hormonal regulation of the immune microenvironment in the mammary gland. J. Mammary Gland Biol. Neoplasia, http://dx.doi.org/10.1007/s10911-014-9324-x. O’Brien, J., Lyons, T., Monks, J., Lucia, M.S., Wilson, R.S., Hines, L., et al., 2010. Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species. Am. J. Pathol. 176, 1241–1255. O’Brien, J., Martinson, H., Durand-Rougely, C., Schedin, P., 2011. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. Development 139, 269–275. Pollard, J.W., Hennighausen, L., 1994. Colony stimulating factor 1 is required for mammary gland development during pregnancy. Proc. Natl. Acad. Sci. U. S. A. 91, 9312–9316. Pradhan, M., Bembinster, L.A., Baumgarten, S.C., Frasor, J., 2010. Proinflammatory cytokines enhance estrogen-dependent expression of the multidrug transporter gene ABCG2 through estrogen receptor and NF{kappa}B cooperativity at adjacent response elements. J. Biol. Chem. 285, 31100–31106. Ramakrishnan, R., Khan, S.A., Badve, S., 2002. Morphological changes in breast tissue with menstrual cycle. Mod. Pathol. 15, 1348–1356. Ratko, T.A., Beattie, C.W., 1985. Estrous cycle modification of rat mammary tumor induction by a single dose of N-methyl-N-nitrosourea. Cancer Res. 45, 3042–3047. Rivas, M.A., Carnevale, R.P., Proietti, C.J., Rosemblit, C., Beguelin, W., Salatino, M., et al., 2008. TNF alpha acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-kappa B-dependent pathways. Exp. Cell Res. 314, 509–529. Rubio, M.F., Werbajh, S., Cafferata, E.G., Quaglino, A., Colo, G.P., Nojek, I.M., et al., 2006. TNF-alpha enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B. Oncogene 25, 1367–1377. Schramek, D., Leibbrandt, A., Sigl, V., Kenner, L., Pospisilik, J.A., Lee, H.J., et al., 2010. Osteoclast differentiation factor rankl controls development of progestin-driven mammary cancer. Nature 468, 98–102. Shi, L., Remer, T., Buyken, A.E., Hartmann, M.F., Hoffmann, P., Wudy, S.A., 2010. Prepubertal urinary estrogen excretion and its relationship with pubertal timing. Am. J. Physiol. Endocrinol. Metab. 299, E990–E997. Tan, W., Zhang, W., Strasner, A., Grivennikov, S., Cheng, J.Q., Hoffman, R.M., et al., 2011. Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling. Nature 470, 548–553. Van Nguyen, A., Pollard, J.W., 2002. Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth. Dev. Biol. 247, 11–25. Varela, L.M., Ip, M.M., 1996. Tumor necrosis factor-alpha: a multifunctional regulator of mammary gland development. Endocrinology 137, 4915–4924. Watson, C.J., 2009. Immune cell regulators in mouse mammary development and involution. J. Anim. Sci. 87, 35–42.
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Hormonal regulation of the cytokine microenvironment in the mammary gland Pallave Dasari a,b,c,1 , David J. Sharkey b,c,1 , Effarina Noordin b,c , Danielle J. Glynn a,b,c , Leigh J. Hodson a,b,c , Peck Y. Chin b,c , Andreas Evdokiou a , Sarah A. Robertson b,c , Wendy V. Ingman a,b,c,∗ a b c
Discipline of Surgery, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Woodville, Australia School of Paediatrics and Reproductive Health, University of Adelaide, Australia Research Centre for Reproductive Health, The Robinson Institute, University of Adelaide, Australia
a r t i c l e
i n f o
Article history: Received 23 April 2014 Received in revised form 4 July 2014 Accepted 5 July 2014 Keywords: Mammary gland Oestrogen Progesterone Cytokines Inflammation
a b s t r a c t The mammary gland is a unique organ that undergoes hormone-driven developmental changes over the course of the ovarian cycle during adult life. Macrophages play a role in regulating cellular turnover in the mammary gland and may affect cancer susceptibility. However, the immune microenvironment that regulates macrophage function has not been described. Hormonal regulation of the cytokine microenvironment across the ovarian cycle was explored using microbead multiplex assay for 15 cytokines in mammary glands from C57Bl/6 mice at different stages of the oestrous cycle, and in ovariectomised mice administered oestradiol and progesterone. The cytokines that were found to fluctuate over the course of the oestrous cycle were colony-stimulating factor (CSF)1, CSF2, interferon gamma (IFNG) and tumour necrosis factor alpha (TNFA), all of which were significantly elevated at oestrus compared with other phases. The concentration of serum progesterone during the oestrus phase negatively correlated with the abundance of cytokines CSF3, IL12p40, IFNG and leukaemia inhibitory factor (LIF). In ovariectomised mice, exogenous oestradiol administration increased mammary gland CSF1, CSF2, IFNG and LIF, compared with ovariectomised control mice. Progesterone administration together with oestradiol resulted in reduced CSF1, CSF3 and IFNG compared with oestradiol administration alone. This study suggests that the cytokine microenvironment in the mammary gland at the oestrus phase of the ovarian cycle is relatively pro-inflammatory compared with other stages of the cycle, and that the oestradiol-induced cytokine microenvironment is significantly attenuated by progesterone. A continuously fluctuating cytokine microenvironment in the mammary gland presumably regulates the phenotypes of resident leukocytes and may affect mammary gland cancer susceptibility. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction ∗ Corresponding author at: Discipline of Surgery, The Queen Elizabeth Hospital DX465702, 28 Woodville Road, Woodville 5011, Australia. Tel.: +61 8 8222 6141; fax: +61 8 8222 6076. E-mail address: [email protected] (W.V. Ingman). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jri.2014.07.002 0165-0378/© 2014 Elsevier Ireland Ltd. All rights reserved.
During the ovarian cycle, the mammary gland epithelium undergoes proliferation, differentiation and apoptosis under the direction of ovarian hormones oestrogen and progesterone (Fata et al., 2001; Ramakrishnan et al., 2002), and this cellular turnover has been linked to the high
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susceptibility of the mammary gland to tumour formation (Ramakrishnan et al., 2002). During the oestrus phase of the cycle, the mammary epithelium exhibits a basic ductal structure. As serum progesterone rises, secondary branching and alveolar bud development occur, with alveolar bud appearance predominantly during dioestrus (Fata et al., 2001). If pregnancy does not proceed, the fall in progesterone at proestrus induces apoptosis of the newly formed alveolar buds, and the tissue is remodelled back to the basic architecture. Macrophages are immune system cells with multiple roles in epithelial cell turnover during the course of the ovarian cycle (Chua et al., 2010). Macrophages promote oestradiol- and progesterone-induced epithelial cell proliferation and alveolar development following ovulation, and when progesterone levels fall towards the end of the cycle, macrophages promote phagocytosis of apoptotic cells and remodel the tissue to its rudimentary structure. These differing macrophage functions are associated with altered phenotype, with fluctuation in expression of MHCII, CD204 and NKG2D by mammary gland macrophages across the cycle under hormonal control (Hodson et al., 2013). In order to recruit macrophages and regulate specific phenotypes and functions of these cells, an array of cytokines and chemokines are likely to be present in the mammary gland microenvironment. Cytokines comprise the interleukins, lymphokines and related regulatory glycoproteins released by cells of the immune system or non-haematopoietic cells, to act as intercellular mediators in the generation of an immune response. Previous studies have shown that cytokines, including colony-stimulating factor 1 (CSF1), interleukin 4 (IL4) and interleukin 13 (IL13), play important roles in mammary gland development during puberty and pregnancy (Pollard and Hennighausen, 1994; GouonEvans et al., 2000; Ingman et al., 2006; Khaled et al., 2007). CSF1, for example, is known to be a regulator of macrophage survival. Csf1 null mutant mice have significantly reduced macrophage abundance and display impaired mammary gland development during puberty and lactation (Gouon-Evans et al., 2000; Ingman et al., 2006). IL4 and IL13 play a role in the promotion of luminal mammary epithelial cell development during pregnancy through the Stat6/Gata3 signalling pathway. Mice with a double mutation in both the Il4 and Il13 genes show a significant decrease in the number of mammary gland side branches and alveolar buds (Khaled et al., 2007). Increased breast cancer risk is associated with early menarche, late menopause and increased cumulative number of menstrual cycles (Chavez-Macgregor et al., 2008; Collaborative Group on Hormonal Factors in Breast Cancer, 2012). Circulating oestrogens are elevated in young women with early menarche (Shi et al., 2010) and this early exposure to oestrogen may be responsible for the increased risk of breast cancer in women with early menarche over and above the increased risk associated with menstrual cycling (Collaborative Group on Hormonal Factors in Breast Cancer, 2012). Fluctuating ovarian hormones presumably create a pro-tumourigenic environment in the
59
mammary gland that, over time, increases the risk of cancer. The biological factors behind this pro-tumourigenic microenvironment have yet to be elucidated, but may include impaired immune surveillance and/or inflammation associated with altered cytokine regulation (Need et al., 2014). The aim of this study was to define the cytokine microenvironment across the four phases of the mouse ovarian cycle and to investigate hormonal regulation of cytokines in the mammary gland. We demonstrate that the concentration of specific cytokines in the mammary gland fluctuates over the course of the ovarian cycle, with the oestrus phase being associated with a relatively pro-inflammatory cytokine microenvironment compared with other stages of the cycle. Furthermore, circulating progesterone negatively correlates with a number of these cytokines at the oestrus phase of the cycle, and in ovariectomised mice progesterone suppresses the oestradiol-induced cytokine profile. Fluctuations in the cytokine microenvironment in the mammary gland over the course of the ovarian cycle may affect susceptibility to mammary gland tumourigenesis. 2. Materials and methods 2.1. Mice All animal experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition, 2004). Mice were maintained in specific pathogen-free conditions with controlled light (12 h light, 12 h dark cycle) and temperature at the Laboratory Animal Services Medical School facility. The oestrous stages of naturally cycling C57Bl/6 mice were determined using cytological characteristics of vaginal smears (Bronson et al., 1968) performed daily. The mice were tracked for a minimum of two weeks through at least one normal four- to five-day oestrous cycle and were 12–15 weeks old at mammary gland dissection. Mice were euthanised at each of the four stages of the oestrous cycle (oestrus, n = 16; metoestrus, n = 20; dioestrus, n = 18; and proestrus, n = 14). At the time of euthanasia, blood was collected by cardiac puncture and the fourth abdominal pair of mammary glands was dissected, snap frozen in liquid nitrogen and stored at −80 ◦ C before protein extraction. To investigate hormonal regulation of the mammary gland cytokine microenvironment, 12-week-old mice were ovariectomised under 2% isoflurane anaesthesia and rested for one week. Subcutaneous 17-oestradiol (1 g) alone or in combination with progesterone (1 mg) was administered in 100 l of sesame oil daily for three days, while control mice were given vehicle alone. On the fourth day, approximately 1 mL of blood was collected by cardiac puncture and the fourth pair of inguinal mammary glands was dissected. The section between the lymph node and the distal end of the gland was snap frozen in liquid nitrogen and stored at −80 ◦ C for cytokine analysis. Blood was centrifuged for six minutes at 350 g and serum was collected and stored at −80 ◦ C.
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2.2. Cytokine analysis Protein was extracted from the mammary gland tissue, using EDTA-free protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA) dissolved in 10 mL of protein extraction buffer containing 500 mM Tris–HCl (Sigma–Aldrich, Castle Hill, NSW, Australia), 200 mM NaCl (Merck, Darmstadt, Germany) and 10 mM CaCl2 (BDH Chemicals, Murarrie, QLD, Australia). The mammary gland was homogenised in 1 mL of the extraction buffer, centrifuged and the aqueous layer collected. After adding 10% Triton X-100 (Bio-Rad Laboratories, Hercules, CA, USA) to the aqueous layer, the solution was homogenised again, the aqueous layer collected and stored at −80 ◦ C. Protein concentration of the sample was measured using Nanodrop ND-100 (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The concentration of cytokines in mammary gland extracts was quantified using Luminex xMAP® technology (Millipore, Billerica, MA, USA), a bead-based multiplex platform for the detection of multiple proteins in a single sample, as per the manufacturer’s instructions. The following cytokines were selected: colony-stimulating factor-1 (CSF1), CSF2, CSF3, interleukin (IL) 1 beta (IL1B), IL2, IL4, IL5, IL6, IL10, IL12p40, IL13, IL17, interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and leukaemia inhibitory factor (LIF). All samples were measured in a single assay. The intra-assay coefficients of variation were less than 10%. The lower detection limits of the assays were 1.1 pg/mL CSF1, 5.6 pg/mL CSF2, 0.9 pg/mL CSF3, 3.2 pg/mL IL1B, 3.2 pg/mL IL2, 0.4 pg/mL IL4, 1.8 pg/mL IL5, 3.3 pg/mL IL6, 3.2 pg/mL IL10, 4.9 pg/mL IL12p40, 3.2 pg/mL IL13, 0.5 pg/mL IL17, 0.9 pg/mL IFNG, 1.0 pg/mL TNFA and 0.8 pg/mL LIF. The concentrations of cytokines measured are presented as picograms per milligramme of protein (pg/mg).
2.3. Serum hormone analysis Oestradiol and progesterone concentrations in serum were measured using commercial RIA kits DSL-4800 and DSL-3400 respectively (Diagnostic Systems Laboratories, Webster, TX, USA) according to the instructions of the manufacturer. All samples were measured in a single assay. The within-assay coefficients of variation were 10.0% in the oestradiol assay and 7.9% in the progesterone assay. The lower limits of detection were 5 pg/mL of oestradiol and 0.3 ng/mL of progesterone.
2.4. Statistical analysis Data were analysed using SPSS Statistics version 20 (IBM Software, New York, NY, USA). The non-parametric Kruskal–Wallis test was conducted, followed by post hoc analysis with Mann–Whitney U test for comparisons of cytokine concentrations among multiple groups. Spearman’s rho test was employed to examine correlations between progesterone and specific cytokines during oestrous phases. Significance was inferred at p < 0.05.
Table 1 Oestradiol and progesterone concentrations in serum during phases of the oestrous cycle in naturally cycling C57/Bl6 mice. Values are mean ± standard error, n = 14–20 per group. Statistics analysis using Kruskal–Wallis test with post hoc Mann–Whitney U test, p < 0.05. Groups assigned ‘a’ differ significantly from groups assigned ‘b’ and ‘c’. Estrous phase
Estradiol (pg/mL)
Oestrus Metoestrus Dioestrus Proestrus
41.3 31.5 34.3 27.3
± ± ± ±
3.3a 2.8ab 3.0ab 1.7b
Progesterone (ng/mL) 1.5 5.9 4.8 2.4
± ± ± ±
0.1a 1.0b 1.2bc 0.5ac
3. Results 3.1. Serum hormone concentration in naturally cycling mice Levels of serum oestradiol and progesterone varied across the phases of the oestrous cycle, as expected (Table 1). Oestradiol peaked during oestrus and was significantly elevated compared with proestrus. Serum progesterone was lowest at the oestrus phase and was significantly increased during metoestrus and dioestrus. 3.2. Fluctuation in cytokine concentration in the mammary gland across the oestrous cycle Cytokine analysis by microbead technology was used to determine the concentration of 15 cytokines in the mammary gland during the oestrous cycle. Of these, IL5, IL6 and IL17 were not detected in the mammary gland at any time during the oestrous cycle (data not shown). The remaining 12 cytokines (CSF1, CSF2, CSF3, IL1B, IL2, IL4, IL10, IL12p40, IL13, IFNG, TNFA and LIF) were all present at detectable levels in mammary gland protein extracts. Since several cytokines showed evidence of fluctuations over the course of the oestrous cycle, the relationship between serum oestradiol and progesterone with mammary gland cytokine concentration was analysed. For all 12 detectable cytokines, no significant correlations with serum oestradiol were detected during any phase (data not shown). During the dioestrus and proestrus phases of the cycle, there were no correlations between cytokines and progesterone (data not shown). However, negative correlations between progesterone and a number of different cytokines were observed during oestrus and metoestrus. The correlations between progesterone and cytokines during the oestrus phase of the cycle were most striking, and are presented for each cytokine in Figs. 1–3. The significant correlations observed at metoestrus are presented in the text below. Concentrations of CSF1 and CSF2 were significantly elevated at oestrus compared with metoestrus, dioestrus and proestrus (Fig. 1A and B respectively). CSF3 concentration did not significantly fluctuate across the oestrous cycle (Fig. 1C). At oestrus, CSF1 and CSF2 had no significant relationship with progesterone (Fig. 1A and B); however, CSF3 negatively correlated with progesterone (rs = −0.55; p = 0.028; Fig. 1C). The concentration of CSF2 showed a negative correlation with progesterone during the metoestrus phase of the cycle (rs = −0.48; p = 0.031).
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A.
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P 4 ( n g /m l) Fig. 1. Colony-stimulating factor (CSF) concentrations in mammary gland of mice. Mammary glands from naturally cycling C57/Bl6 mice were harvested at different phases of the oestrous cycle and protein extracted to measure with Luminex xMAP Technology (A) CSF1, (B) CSF2 and (C) CSF3, as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) CSF1, (B) CSF2 and (C) CSF3, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
Six interleukins (IL1B, IL2, IL4, IL4, IL12p40 and IL13) were detected continuously over the course of the oestrous cycle, with no significant variation between phases (Fig. 2). However, mammary gland IL-12p40 concentration exhibited a significant negative correlation with progesterone during the oestrus phase of the cycle (rs = −0.54; p = 0.033).
At metoestrus, progesterone negatively correlated with IL2 (rs = −0.65; p = 0.002), IL4 (rs = −0.51; p = 0.022), IL10 (rs = −0.50; p = 0.025), IL12p40 (rs = −0.47; p = 0.038). The concentration of IFNG was significantly increased at oestrus compared with metoestrus, dioestrus and proestrus (Fig. 3A). TNFA concentration was significantly
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Fig. 2. Interleukin (IL) concentrations in the mammary glands of mice. Interleukin concentrations measured in mammary glands from naturally cycling C57/Bl6 mice were harvested during different phases of the oestrous cycle; (A) IL1B, (B) IL2, (C) IL4, (D) IL10, (E) IL12p40 and (F) IL13, as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) IL1B, (B) IL2, (C) IL4, (D) IL10, (E) IL12p40 and (F) IL13, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
higher during oestrus compared with dioestrus and proestrus (Fig. 3B). LIF concentration did not significantly fluctuate across the cycle (Fig. 3C). IFNG and LIF concentrations show negative correlation with progesterone during the oestrus phase of the cycle (rs = −0.56; p = 0.025 and rs = −0.57; p = 0.023 respectively). 3.3. Hormonal regulation of mammary gland cytokines Circulating progesterone appeared to have a profound negative regulatory effect on mammary gland cytokine concentration in naturally cycling mice. This was particularly evident during the oestrus phase of the cycle when circulating oestradiol peaks, and the concentration of a number of cytokines are increased. This suggests that oestradiol induces production of these cytokines, while progesterone dampens this effect. To investigate the effect of ovarian hormones on mammary gland cytokine concentration, ovariectomised mice were treated with vehicle control, oestradiol only, or oestradiol together with progesterone. Further microbead analysis of cytokines was
conducted on those cytokines shown to fluctuate across the natural cycle or to be negatively regulated by progesterone during the oestrus phase. Mammary gland CSF1 concentration in oestradioltreated mice was significantly higher than in control mice and in those treated with oestradiol and progesterone (Fig. 4A). In contrast, CSF2 concentration was significantly lower in mammary glands from oestradiol-treated mice than in those from mice treated with oestradiol and progesterone (Fig. 4B). CSF3 concentration was decreased in mammary glands from mice treated with oestradiol and progesterone compared with oestradiol-treated mice (Fig. 4C). The concentration of IFNG in mammary glands from oestradiol-treated mice was significantly elevated compared with those from control mice and mice treated with oestradiol and progesterone (Fig. 4E). Concentration of LIF was higher in oestradiol-treated mice than in control mice (Fig. 4G). There was no significant effect of oestradiol or progesterone on IL12p40 (Fig. 4D) or TNFA (Fig. 4F) concentration in mammary gland protein extracts.
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A. 0 .5
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P 4 ( n g /m l) Fig. 3. Proinflammatory cytokine concentrations in the mammary glands of mice. Cytokine concentrations measured in mammary glands from naturally cycling C57/Bl6 mice were harvested during different phases of the oestrous cycle: (A) interferon gamma (IFNG), (B) tumour necrosis factor (TNFA) and (C) leukaemia inhibitory factor (LIF), as seen in the graphs on the left. Progesterone concentrations during oestrus correlated with (A) IFNG, (B) TNFA and (C) LIF, as seen in the graphs on the right. Bars represent mean values, n = 14–20 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups. Spearman’s rho test was used to assess correlation (*p < 0.05).
4. Discussion A number of cytokines have been ascribed important roles in the events of mammary gland development and remodelling during puberty, pregnancy, lactation and involution (Pollard and Hennighausen, 1994; Gouon-Evans et al., 2000; Ingman et al., 2006; Khaled et al., 2007; Ingman
and Robertson, 2008; Watson, 2009; O’Brien et al., 2010). However, the abundance and types of cytokines in the adult cycling mammary gland have not been investigated. In part this is because the gland is relatively quiescent in non-pregnant adult females, compared with the extensive morphogenesis that occurs during puberty, pregnancy and involution. However, there are indeed structural and
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A.
*
150
E.
*
*
50
*
IF N G ( p g /m g )
C S F 1 ( p g /m g )
40 100
50
0 C
*
1 .5
E2 + P4
F.
*
1 .0
0 .5
E2
C
E2
E2 + P4
E2
E2 + P4
0 .2
*
G. 0 .1 0
*
50
0 .0 8
L IF ( p g /m g )
C S F 3 ( p g /m g )
E2 + P4
0 .4
E2 + P4
40
0 .0 6
30
0 .0 4
20
0 .0 2
10
0 .0 0
0
IL 1 2 p 4 0 ( p g /m g )
E2
0 .0 C
D.
C
0 .6
T N F A ( p g /m g )
C S F 2 ( p g /m g )
E2
0 .0
C.
20 10
0
B.
30
C
E2
E2 + P4
C
E2
E2 + P4
C
0 .5 0 .4 0 .3 0 .2 0 .1 0 .0
Fig. 4. Cytokine production in response to hormonal regulation. C57/Bl6 mice were ovariectomised and treated with control, oestradiol (E2) or oestradiol and progesterone (E2 + P4) for three days before mammary glands were harvested and measured (A) CSF1, (B) CSF2, (C) CSF3, (D) IL12p40, (E) IFNG, (F) TNFA and (G) LIF. Bars represent mean values, n = 7–12 per group. Kruskal–Wallis test with post hoc Mann–Whitney U test to compare groups (*p < 0.05).
P. Dasari et al. / Journal of Reproductive Immunology 106 (2014) 58–66
immunological changes that accompany each phase of the oestrous cycle. In particular, previous studies in mice have demonstrated that macrophage phenotype and function fluctuate across the ovarian cycle, and are regulated by the ovarian hormones oestradiol and progesterone (Chua et al., 2010; Hodson et al., 2013), suggesting that there might be accompanying changes in the cytokine microenvironment. In this study we provide evidence of fluctuating cytokine activity in the mammary gland microenvironment throughout the ovarian cycle. IFNG, TNFA, CSF1 and CSF2 concentrations were elevated during oestrus when circulating oestradiol peaked, and declined during metoestrus, dioestrus and proestrus. Although exogenous oestradiol administration caused elevation in a number of cytokines in ovariectomised mice, cytokine abundance did not correlate with circulating oestradiol in naturally cycling mice. We observed substantial variation in cytokine abundance during oestrus between mice, and a number of mice analysed at this stage of the cycle did not exhibit elevated cytokine concentration. Further analysis of this variation at oestrus revealed significant negative correlations between circulating progesterone and mammary gland cytokine concentration of CSF3, IL12, IFNG and LIF during this phase of the cycle. Moreover, administration of exogenous progesterone attenuated oestradiol-induced elevation in cytokine abundance in ovariectomised mice. This suggests that although oestradiol induces cytokine production, progesterone has a strong suppressive effect on these specific cytokines, even at the low circulating level of progesterone observed during the oestrus phase of the cycle. Therefore, we propose that the fluctuation in cytokine concentration in the mammary gland across the cycle is due to induction of cytokine production by oestradiol during the oestrus phase of the cycle, which is suppressed by progesterone during the other phases. Results from previous studies suggest that cytokine activity in the mammary gland tends to be associated with active periods of macrophage-assisted morphogenesis. CSF1 is a chemoattractant for macrophages (Van Nguyen and Pollard, 2002) and promotes mammary morphogenesis during puberty, pregnancy and involution (Pollard and Hennighausen, 1994; Gouon-Evans et al., 2000; Ingman et al., 2006; O’Brien et al., 2011). CSF2 is also important in regulating macrophage function, proliferation and survival (Lehtonen et al., 2002). IFNG and TNFA expression in the mammary gland is elevated during pregnancy and decreased during lactation (Varela and Ip, 1996; Khaled et al., 2007). LIF mRNA has been shown to increase in the mammary gland during involution, and may be necessary for regression during early involution by initiating epithelial cell apoptosis (Clarkson et al., 2004). As dioestrus is the most active phase of the cycle in respect of epithelial cell activity (Fata et al., 2001) and macrophage abundance (Chua et al., 2010), with the oestrus phase considered relatively inactive, the elevation in cytokines during the oestrus phase of the cycle and by exogenous oestradiol administration was surprising. The roles of these cytokines in mammary gland development and function at the oestrus phase are unclear; however, our results indicate a highly active immune microenvironment at this time. One possibility is that the elevated cytokines
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associated with oestrus initiate pathways of immune adaptation, which in the event of conception progress to the further morphogenesis and functional changes seen in pregnancy. We have not investigated the cell types in the mammary gland responsible for cytokine synthesis; however, epithelial cells, fibroblasts, adipocytes and immune cells, including macrophages, are all capable of oestradiolinduced cytokine secretion, and may contribute to this cytokine microenvironment. Our results suggest that the pro-inflammatory cytokines TNFA, IFNG and LIF may have under-appreciated roles in the mammary gland. Further studies are required to understand how these cytokines contribute to mammary gland morphogenesis and homeostasis. In many tissues, elevated pro-inflammatory cytokine concentrations are linked to tumour promotion and progression (Karin and Greten, 2005). Pro-inflammatory cytokines, including TNFA and CSF1, can stimulate macrophages to produce nitric oxide and reactive oxygen species, causing DNA damage and mutations. Activation of the NF-kB signalling pathway by TNFA causes elevation in the abundance of pro-inflammatory cytokines, such as IFNG, leading to tumour promotion and progression (Karin and Greten, 2005; Lin and Karin, 2007; Rivas et al., 2008). TNFA, in synergy with oestradiol, promotes the proliferation of breast cancer cells (Rubio et al., 2006) and drug-resistant breast cancer tumours (Pradhan et al., 2010). CSF1 also contributes to excessive inflammatory responses by regulating macrophage function to produce more proinflammatory cytokines (Irvine et al., 2006). Therefore, the increased abundance of these pro-inflammatory cytokines during oestrus may promote a tumourigenic environment. On the other hand, progesterone-regulated immune cells may also have an impact on breast cancer risk. Carcinogenic effects of progesterone are mediated locally within the mammary gland epithelium by the tumour necrosis factor-related cytokine RANK ligand (GonzalezSuarez et al., 2010; Schramek et al., 2010). Production of RANK ligand by infiltrating regulatory T cells promotes mammary cancer progression (Tan et al., 2011), and inhibition of this pathway might be utilised to prevent breast cancer (Gonzalez-Suarez et al., 2010). Progesterone withdrawal appears to inhibit tumour immune surveillance (Hodson et al., 2013), which might be associated with the increased risk of mammary cancer observed in rats injected with chemical carcinogen during the proestrus phase of the ovarian cycle, compared with the dioestrus phase (Ratko and Beattie, 1985). Thus, it seems that both extremes of the hormone fluctuations associated with the ovarian cycle raise the risk of tumour induction and progression through inflammation driving tumourigenesis and through altered immune surveillance. 5. Conclusion Here we report that the cytokine microenvironment in the mammary gland fluctuates over the course of the ovarian cycle and is hormonally regulated. A number of pro-inflammatory cytokines are elevated during the oestrus phase of the cycle. Administration of exogenous
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