<p>Chemokines in the Female Reproductive Tract </p><p>Laboratory Report</p><p>Carolann Waddell Word count: 1993 </p><p>Abstract Introduction: Inflammatory responses are associated with reproductive processes. Chemokines help to regulate these inflammatory pathways by mediating leukocyte recruitment into reproductive tissues. The purpose of this study was to examine the expression of the chemokines CXCL1, 2, 3 and 5 in the vagina and uterine horn of CXCR2-/- and CXCR2 +/+ mice and also to determine CCL27 protein expression in the ovary of C57/BL6 mice. </p><p>Methods: C57BL/6, BALB/C and CXCR2 -/- (BALB/C background) mice were used in experiments. mRNA expression of CXCL1, 2, 3 and 5 was determined using qRT-PCR and CCL27 protein expression was determined by ELISA. </p><p>Results: There were no statistically significant differences between expression of the CXC chemokines in the vagina or uterine horn of CXCR2-/- compared with CXCR2+/+ mice. CCL27 protein was highly expressed in the C57BL/6 mouse ovary, and was approximately 10-fold higher than CCL27 protein concentration in the skin, uterine horn and vagina, p<0.05. </p><p>Conclusions: CXC chemokine expression in the murine vagina and uterine horn suggests a role in the recruitment of neutrophils to these sites. CCL27, which is involved in memory T cell recruitment, may function to modulate T cell trafficking to the ovary. The presence and trafficking of neutrophils and T cells to the vagina and ovary respectively, warrants further investigation. </p><p>Introduction </p><p>2 Almost all female reproductive processes including ovulation, menstruation, implantation, placentation, labour and post partum remodelling are associated with inflammatory responses. [1, 2] Depending on the tissue and process involved, a unique repertoire of immune cells, pathways and inflammatory mediators are expressed. They function to initiate and execute reproductive processes as well as to repair and remodel reproductive tissues to preserve future reproductive function. This occurs in a cyclical fashion throughout a female’s reproductive life cycle under the influence of the ovarian steroid hormones progesterone and oestrogen. Locally, inflammatory responses are controlled and regulated by a host of inflammatory mediators such as prostaglandins, growth factors, cytokines and chemokines. Importantly, aberrant inflammatory pathways are thought to contribute to a variety of pathologies of female reproductive function such as polycystic ovarian syndrome (PCOS), ovarian cancer, premature ovarian failure, unexplained infertility, recurrent miscarriage, endometriosis, pre-eclampsia and preterm labour. [1]</p><p>Chemokines are small polypeptides, typically 8-14kDa in length. They are secreted by a variety of cells and are primarily involved in recruitment of leukocytes into tissues. Approximately 50 human chemokines have been identified. They bind to G-coupled transmembrane receptors, of which around 19 are known. [3] Generally one chemokine will bind many receptors and one receptor will bind many chemokines, making it difficult to define their specific roles. To complicate matters further it is likely that the sequential or combinatorial action of multiple chemokines is necessary for the recruitment of specific populations of immune cells. Moreover it is now recognised that chemokines have non-immune functions such as promoting angiogenesis, differentiation and proliferation in tissues. [3] A number of studies have identified important physiological roles for chemokines in the reproductive tract. [2] </p><p>Recent work in our laboratory (unpublished) found that the CXC chemokines CXCL1, 2, 5 and 7 were highly expressed in the cervix and vagina of C57BL/6 3 Carolann Waddell 28 November 2012 mice compared to other solid tissues. This also corresponded with high levels of Neutrophil Granule Protein (NGP) in these tissues, a known neutrophil marker. Neutrophils are the predominant CXCR2+ leukocytes in peripheral blood and CXCL1, 2, 5 and 7 mediate neutrophil recruitment through activation of this receptor. In addition, we also found that mRNA levels of the chemokine, CCL27 were high in the ovary. This was a notable finding because other chemokines examined had low levels of expression in this tissue type. </p><p>Thus, the aim of this study was to investigate the role of the CXC chemokines in the female reproductive tract, by comparing the expression of CXCL1, 2, 5 and 7 in CXCR2 +/+ and CXCR2 -/- mice using qRT-PCR. We also aimed to confirm the presence of CCL27 protein in the ovary, and quantify this expression by ELISA, using lysates of cervix, ovary, skin, uterine horn and vagina collected from C57/BL6 mice. </p><p>Methods </p><p>4 Animals Animal work was carried out under UK Home Office licenses. C57BL/6 mice, BALB/C mice and CXCR2 -/- (BALB/C background) mice were maintained in specific pathogen-free conditions at the University of Glasgow’s Central Research Facility. Stage of estrus cycle was determined by observation of vaginal appearance and by microscopic examination of cell types within the vaginal washout. </p><p>Number at each stage of estrus cycle in each strain of mouse used Proestrus Estrus Metestrus Diestrus BALB/C CXCR2 +/+ (n=5) / 4 1 / BALB/C CXCR2 -/- (n=7, 1 N/A) 4 2 / / C57BL/6 (n=12) 2 2 4 4</p><p>Table 1. Numbers of mice from each strain at each stage of the estrus cycle when tissues were collected. </p><p>RNA extraction Cervix, ovary and skin were removed from BALB/C and CXCR2-/- (BALB/C background) mice, snap frozen in liquid nitrogen and stored at -80 oC until required for RNA extraction. Mouse tissue was homogenised in 1ml of Trizol (Invitrogen) using an Omni µH homogenizer and total RNA was isolated according to the manufacturer’s instructions. Quantity of RNA (ng/ul) and 260/280 absorbance of each RNA preparation were measured using Nanodrop nd-100 spectrophotometer (Nanodrop Technologies Inc.). </p><p>Preparation of cDNA 5µg of RNA was DNAase treated using the DNA-freeTM kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. cDNA was synthesised from DNAase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Warrington, UK) according to manufacturer’s instructions. cDNA was stored at -20oC until required. </p><p>Quantitative Real-Time PCR (qRT-PCR) qRT-PCR was performed using the ABI Prism 7900HT system (Applied Biosystems). Each reaction contained 12.5ul of 2x Taqman mastermix</p><p>5 Carolann Waddell 28 November 2012 (Applied Biosystems), 10.25ul of DEPC-treated water, 1.25ul of 20x target assay or endogenous control assay (Applied Biosystems) and 1ul of sample cDNA. One microliter of 1:10 dilution of cDNA was added to each well that included the endogenous control gene, GAPDH. Probes and control assays used are shown in Table 2. The thermal cycler conditions were 50 oC for 2 minutes, 95oC for 10 minutes then 40x 95oC for 15 seconds and 60oC for 1 minute. Data was analysed using sequence detection software 2.3, which calculated the threshold cycle (Ct) values. Target gene expression was normalised by subtracting the Ct value of GAPDH, the endogenous control, from the Ct value of the target assay. Fold increase relative to the control was obtained using the formula 2-ΔCt and expressed as a percentage.</p><p>Applied Biosystems NCBI reference Gene Gene symbol assay ID sequence Mouse CXCR2 Cxcr2 Mm99999117_s1 NM_009909.3 Mouse CXCL1 Cxcl1 Mm00433858_g1 NM_008176.3 Mouse CXCL2 Cxcl2 Mm00436450_m1 NM_009140.2 Mouse CXCL5 Cxcl5 Mm00436451_g1 NM_009141.2 Mouse CXCL7 Ppbp Mm01347901_g1 NM_023785.2 Mouse GAPDH Gapdh Mm99999915_g1 NM_008084.2</p><p>Table 2. Target assay mixes and endogenous control probes used in qRT-PCR </p><p>Collection of protein lysates Cervix, ovary, skin, uterine horn and vagina were removed from C57BL/6 mice, snap frozen in liquid nitrogen and stored at -80oC until required for protein lysate preparation. Tissues were weighed and 400µl of Cell Lytic MT solution (Sigma) containing protease inhibitors (Roche) added to each tissue sample. Tissues were homogenised and lysates cleared of debris by centrifugation at 13000rpm for 10 minutes at 4oC. Supernatant containing protein was collected and stored at -20oC until required. </p><p>ELISA CCL27 concentration of each sample was measured using a DuoSet ELISA kit (R&D systems) according to the manufacturer’s instructions. Optical density of each sample was measured at 450nm (with correction) and</p><p>6 analysed on a microplate-reader. CCL27 concentration is presented as amount of chemokine (pg) per mg of tissue.</p><p>Statistical analysis Data was analysed using GraphPad Prism version 5.04 software. Mann- Whitney U-test or Kruskal-Wallis test (with Dunn’s multiple comparison test) was performed as appropriate, p < 0.05 was accepted as significant. </p><p>Results </p><p>Expression of CXC chemokines in female reproductive tissues</p><p>Despite previous studies in C57BL/6 mice demonstrating that the CXC chemokines were highly expressed in both the cervix and vagina, only uterine</p><p>7 Carolann Waddell 28 November 2012 horn and vaginal tissues from CXCR2-/- and CXCR2+/+ were available for analysis. Cervical tissue from these mice is currently being collected. There were no statistically significant differences between expression of the CXC chemokines in the vagina or uterine horn of CXCR2-/- compared with CXCR2+/+ mice (Fig 1). </p><p>CXCL2 UHORN CXCL5 UHORN CXCL7 UHORN CXCR2 UHORN CXCL1 UHORN 1.0 2.5 15 8 E E 1.5 E E S S S</p><p>E S</p><p>- - - / S - / /</p><p>/ +</p><p>- 2.0 + +</p><p>0.8 + /</p><p>) 6 ) ) ) +</p><p>% ) % % % ( ( ( 10</p><p>(</p><p>%</p><p> n ( n 1.0 1.5 n n 0.6 o o o i o i i n i s s s o s 4 i s s s s s e e e e r s r r r 0.4 1.0 p e p p p r x x x x 5 p E E E E</p><p> x</p><p>0.5 e</p><p> e 2 E e e</p><p> v v v v i i</p><p>0.5 i i 0.2 e t t t t v a a a a i l l l l t e e e e a l R R R R 0.0 e 0.0 0 0 R 0.0</p><p>CXCR2 VAGINA CXCL1 VAGINA CXCL2 VAGINA CXCL5 VAGINA CXCL7 VAGINA 15 6 80 2500 80 E E E E E S S S S</p><p>S</p><p>- - - - / / / - / / + + + +</p><p>+ 2000</p><p>) ) 60 ) 60 ) ) % % % % ( ( 4 % (</p><p>( 10</p><p>(</p><p> n n n n n</p><p> o o 1500 o o i i i i o i s s s s</p><p>40 s 40 s s s s s e e e e r r e r r r p p p p 1000 p x x x x</p><p>5 2 x E E E E</p><p>E</p><p> e e 20 e 20 e e v v v v i i i i v t t t t i 500 t a a a a l l l l a l e e e e e R R R R 0 0 0 R 0 0</p><p>Figure 1. qRT-PCR analysis of CXC chemokine (CXCL1, 2, 5 and 7) expression in vagina/ uterine horn in CXCR2-/- (n=7) compared with CXCR2+/+ (n=5) mice. Expression was normalised to GAPDH. Data is presented as mean +/- SEM. No statistically significant differences in chemokine expression were found between CXCR2-/- and CXCR2 +/+ mice, as determined by Mann-Whitney U-test. </p><p>Quantification of CCL27 protein in female reproductive tissues</p><p>CCL27 protein was highly expressed in the mouse ovary, and was approximately 10-fold higher than CCL27 protein concentration in the skin, uterine horn and vagina, p<0.05 (Fig 2). </p><p>8 CCL27 * * 150 * *</p><p> e u s s</p><p> i 100 t</p><p> f o</p><p> g m / g</p><p> p 50</p><p>0</p><p>Figure 2. CCL27 protein in lysates of cervix, ovary, skin, uterine horn and vagina from C57/BL6 mice (n=12), measured by ELISA. Data is presented as mean +/- SEM. *p<0.05, Kruskal-Wallis test with Dunn’s Multiple Comparison Test. </p><p>Discussion </p><p>Previous studies, which utilised C57BL/6 mice, found the expression of CXCL1, 2, 5 and 7 to be high in the vagina and cervix. In addition, expression of these chemokines was influenced by estrus cycle stage with all</p><p>9 Carolann Waddell 28 November 2012 chemokines showing highest levels of expression during the metestrus and diestrus stages. The CXCR2-deficient mice available for use were on a different mouse background (BALB/c) to those used in previous studies. Different strains of mice could potentially express different repertoires of chemokines, so studies should be repeated in both strains. The primary aim of this study presented, was to determine the effect that a deficiency in expression of CXCR2, the receptor for CXCL 1, 2, 5 and 7, has on the chemokine profile within the female reproductive tissues. We found no statistical differences in expression of CXCL1, 2, 5, and 7 in the vagina or uterine horn of CXCR2-/- and CXCR2+/+ mice. </p><p>Although stage of estrus cycle in all mice was determined, we did not take account of this in our analysis because numbers in each group would be too small to yield reliable results. This could explain the large spread of data in each group and why we were unable to detect significantly statistical differences between the CXCR2-/- and CXCR2+/+ mice. We aim to include stage of estrus cycle in our analysis once sufficient numbers of mice have been obtained. This will allow us to understand the impact that the estrus cycle has on the expression profile of these chemokines, and may give some insight into their functions in the vagina and uterine horn. </p><p>Future studies should confirm protein expression of CXCL 1, 2, 5 and 7 in the reproductive tissues because mRNA and protein expression do not always correlate. Chemokine protein expression could also be localised using immunohistochemistry in order to characterise the distribution of these chemokines in these tissues. CXCL 1, 2, 5 and 7 are known to mediate neutrophil recruitment via CXCR2. Using immunohistochemistry, we plan to localise and quantify neutrophils in reproductive tissues and compare differences in number and distribution between CXCR2-/- and CXCR+/+ mice. This will provide insight regarding the role of CXCR2 in recruitment of neutrophils to reproductive tissues, the vagina in particular. The vagina is a mucosal tissue and is therefore exposed to the external environment. Neutrophil recruitment to this site is essential in order to provide immunological protection against potential pathogens. </p><p>10 In addition to studying the CXC chemokines, we wished to further study the CC chemokine, CCL27. We found CCL27 protein to be highly expressed in the mouse ovary compared to other tissues examined, consistent with our previous findings in which high levels of CCL27 mRNA were expressed in this tissue. Future studies will examine the spatial distribution of this protein in the murine ovary using immunohistochemistry. Estrus cycle stage was not accounted for in these studies as CCL27mRNA did not exhibit differential expression, however, as previously mentioned stage of estrus cycle can influence chemokine abundance and distribution. This will also be a focus of future experiments. </p><p>The function of CCL27 in the ovary is unknown; however the high abundance we observed in our study suggests an important role in ovarian physiology. During skin inflammation CCL27 mediates memory T-cell recruitment via CCR10. [4] With this in mind CCL27 could be involved in T-cell trafficking to the ovary. Memory T cells have been found to be significantly reduced in the ovaries of women with PCOS compared to non-PCOS ovaries, implicating memory T cells in the pathogenesis of the disease. [5] Thus, understanding the role of CCL27 in T-cell trafficking to the ovary could also have important implications for understanding ovarian pathology in humans. </p><p>There is increasing evidence implicating chemokines in reproductive processes. They are also thought to be involved in the pathogenesis of many diseases of the reproductive system, making them candidates for therapy. However, the repertoire of chemokines involved, and the function they perform has not been fully characterised. We have shown mRNA expression of CXCL 1, 2, 5 and 7 in the murine vagina and uterine horn as well CCL27 mRNA and protein expression in the murine ovary. Further work is required to depict the role of these chemokines in these tissues and their contributions to reproductive processes. However, when interpreting these results it is important to note that this is a snapshot of a complex dynamic biological system. It is likely that the sequential or combinatorial action of multiple</p><p>11 Carolann Waddell 28 November 2012 chemokines is necessary for the recruitment of specific populations of immune cells and to drive specific inflammatory pathways.</p><p>References </p><p>1. Jabbour HN, Sales KJ, Catalano RD, Norman JE. Inflammatory pathways in female reproductive health and disease. Reproduction. [Online] 2009; 138(6): pp 903-19. Available from: doi: 10.1530/REP-09-0247 [Accessed November 2012] </p><p>12 2. Kitaya K, Yamada H. Pathophysiological roles of chemokines in human reproduction: an overview. Am J Reprod Immunol. [Online] 2011; 65(5): pp 449-59. Available from: doi: 10.1111/j.1600-0897.2010.00928.x. [Accessed November 2012]</p><p>3. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. [Online] 2006; 354 (6): pp 610-21. Available from: doi: 10.1056/NEJMra052723 [Accessed November 2012]</p><p>4. Homey B, Alenius H, Muller A, Soto H, Bowman EP, Yuan W, McEvoy L, Lauerma Al, Zlotnik A, et al. CCL27-CCR10 interactions regulate T-cell mediated skin inflammation. Nat Med. [Online] 2002 8(2): pp 157-65 Available from: doi: 10.1038/nm0202-157 [Accessed November 2012] </p><p>5. Wu R, Fujii S, Ryan NK, Van der Hoek KH, Jasper JM, Sini I, Robertson SA. Ovarian leukocyte distribution and cytokine/chemokine mRNA expression in follicular fluid cells in women with polycystic ovary syndrome. Hum Reprod. [Online] 2007 22(2): pp 527-35 Available from: doi: 10.1093/humrep/del371 [Accessed November 2012] </p><p>13 Carolann Waddell 28 November 2012</p>