Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation

Molecular Human Reproduction, May 2009

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs), present in epithelial cells, monocytes and dendritic cells. This study details their expression, regulation and role in human endometrium. Real-time PCR showed that NOD1 mRNA is constitutively expressed in endometrium. NOD2 is up-regulated in the late secretory phase of the menstrual cycle suggesting a role in menstruation. Both proteins are immunolocalized in endometrial epithelium, stroma and endothelium. In first trimester, decidua NODs are present in decidualized stroma. NOD function was examined in endometrial stromal cells (ESCs) and endometrial epithelial cells (EEpCs) in vitro. IκBα is up-regulated by stimulation of ESC and EEpC with an NOD1 ligand. IκBα, IL-8 and TNFα mRNA expression is increased in EEpC by a NOD2 ligand. NOD2 mRNA expression increases in response to IL-1 treatment while NOD1 transcripts are unaltered. NOD1 mRNA is increased in an in vitro model of decidualization of ESC. In summary, we report expression of NOD1 and NOD2 in human endometrium and show that they are differentially regulated. NOD2 and, to a lesser extent, NOD1 can function to increase expression of innate immune molecules in endometrium. NODs may have a role in innate immune protection in the uterus and NOD2 may regulate inflammation associated with menstruation.

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Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation

Anne E. King 2 Andrew W. Horne 2 Sabine Hombach-Klonisch 0 1 J. I. Mason 2 Hilary O.D. Critchley 2 0 Department of Obstetrics and Gynecology, University of Manitoba , Winnipeg , Manitoba, Canada 1 Department of Human Anatomy and Cell Science, University of Manitoba , Winnipeg , Manitoba, Canada 2 Reproductive and Developmental Sciences, Centre for Reproductive Biology, The Queen's Medical Research Institute , 47 Little France Crescent, Edinburgh EH16 4TJ, UK Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs), present in epithelial cells, monocytes and dendritic cells. This study details their expression, regulation and role in human endometrium. Real-time PCR showed that NOD1 mRNA is constitutively expressed in endometrium. NOD2 is up-regulated in the late secretory phase of the menstrual cycle suggesting a role in menstruation. Both proteins are immunolocalized in endometrial epithelium, stroma and endothelium. In first trimester, decidua NODs are present in decidualized stroma. NOD function was examined in endometrial stromal cells (ESCs) and endometrial epithelial cells (EEpCs) in vitro. IkBa is up-regulated by stimulation of ESC and EEpC with an NOD1 ligand. IkBa, IL-8 and TNFa mRNA expression is increased in EEpC by a NOD2 ligand. NOD2 mRNA expression increases in response to IL-1 treatment while NOD1 transcripts are unaltered. NOD1 mRNA is increased in an in vitro model of decidualization of ESC. In summary, we report expression of NOD1 and NOD2 in human endometrium and show that they are differentially regulated. NOD2 and, to a lesser extent, NOD1 can function to increase expression of innate immune molecules in endometrium. NODs may have a role in innate immune protection in the uterus and NOD2 may regulate inflammation associated with menstruation. Introduction The human endometrium is a dynamic tissue which undergoes repeated cycles of proliferation, differentiation and regeneration (Jabbour et al., 2006). Both implantation and menstruation are associated with a physiological inflammatory response in the endometrium when increased production of cytokines and infiltration of leukocytes occurs under the control of the sex steroid hormones, estradiol and progesterone and locally produced inflammatory mediators (Jabbour et al., 2006). The endometrium is capable of supporting a developing embryo while, in common with all mucosal surfaces, maintaining the ability to respond to infection. Genital tract infection is associated with infertility, ectopic pregnancy and preterm labour and the innate immune response is an important component of the mucosal defence system (King et al., 2003a; Horne et al., 2008). Pattern recognition receptors (PRRs) recognize pathogenassociated molecular patterns (PAMPs) allowing them to detect infection and initiate the innate immune response, resulting in increased production of inflammatory cytokines, natural antimicrobials and stimulation of the adaptive immune response (Mitchell et al., 2007; Carneiro et al., 2008). Toll-like receptors (TLRs) are cell surface PRRs that respond to a wide range of PAMPs, including those produced by bacteria, viruses and fungi (Mitchell et al., 2007). TLRs have been reported to be widely expressed in the female reproductive & The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: Table I Details of endometrial biopsies 455.3 (203.76 599.12) 638.4 (276 1949) 172.1 (59.09 819) 72.4 (5.5 112.91) 83.5 (42.10 114.53) 5.4 (1.06 11.29) Cycle phase (as determined by the Noyes criteria) was consistent with circulating estradiol and progesterone concentrations. PROL, proliferative; ES, MS and LS, early, mid and late secretory; MENST, menstrual. tract (Fazeli et al., 2005). They are present in human endometrium and are expressed in a cycle-dependent manner (Aflatoonian et al., 2007; Hirata et al., 2007). We have previously shown that innate immune effector molecules, including b-defensins and elafin, show similar cycle-dependent expression (King et al., 2000, 2003b, c; Fleming et al., 2003). These findings suggest that the endometrial innate immune response is modulated by ovarian steroids. Nuclear oligomerization domain (NOD) proteins are cytosolic PRRs. NOD1 and NOD2 detect peptidoglycans from bacteria: NOD1 detects meso-diaminopimelic acid (iE-DAP) present mainly on Gram-negative bacteria while NOD2 responds to muramyl dipeptide (MDP) found on both Gram-negative and -positive pathogens (Chamaillard et al., 2003; Girardin et al., 2003a, b). Both NODs direct the innate immune response by activation of the NFkB pathway (Inohara et al., 2001; Ogura et al., 2001). NOD1 and NOD2 are present in monocytes/macrophages, dendritic and epithelial cells (Ogura et al., 2001; Gutierrez et al., 2002; Fritz et al., 2005; Uehara et al., 2007). There are few reports detailing expression of NOD1 and NOD2 in the female reproductive tract. While both proteins have been detected in the trophoblast of first trimester pregnancy (Costello et al., 2007), there are no reports examining their expression in human endometrium. The aim of the current study is to investigate the expression, regulation and function of NOD1 and NOD2 in human endometrium. Methods Tissue collection Endometrium (n 31) was collected from women (median 40; age range 28 48) undergoing procedures for benign gynaecological conditions including menstrual complaints, uterine prolapse or prior to the insertion of intrauterine contraception. All women had regular menstrual cycles (25 35 days) and had not undergone hormonal treatment in the 3 months preceding biopsy. All biopsies were histologically dated using the Noyes criteria and these datings corresponded with circulating serum estradiol and progesterone concentrations (see Table I). First trimester decidua (n 8; 8 12 weeks) was collected from women undergoing surgical termination of pregnancy. A viable intrauterine pregnancy and gestational age were confirmed by ultrasound scan. At the time of collection, tissue biopsies were: (i) fixed in neutralbuffered formalin (4%) for 24 h, stored in 70% ethanol and wax embedded; (ii) immersed in RNAlater (Ambion, Austin, TX, USA) for subsequent RNA extraction; and/or (iii) used for endometrial stromal cell isolation (see below). Written informed consent was obtained from all patients and ethical approval was granted by the Lothian research ethics committee. Quantitative RT PCR RNA was extracted from cells/tissues as detailed in the manufacturers protocol (Qiagen, RNeasy mini kits). All samples were treated with DNaseI (Qiagen) in order to remove any contaminating genomic DNA. Quantitative real-time RT PCR was used to measure mRNA levels of NOD1 and NOD2. Complementary DNA was prepared in 20 ml reaction volumes containing: 10 RT buffer (1 ), magnesium chloride (5.5 mM), dNTP mix (2 mM), random hexamers (2.5 mM), RNase inhibitor (0.4 U/ml) and Multiscribe reverse transcriptase (1.25 U/ml; Applied Biosystems, Warrington, Cheshire, UK). 200 ng of template RNA were reverse transcribed and each cDNA experiment included two controls: one containing template RNA but no reverse transcriptase (RT negative) and the other containing reverse transcriptase with water in place of template RNA (RT H2O). PCR mixtures contained Taqman 2 Master-mix (1 ; Applied Biosystems), forward and reverse primers (300 nM; Eurogentec) and probe (200 nM; Eurogentec) for NOD1 or NOD2 and forward and reverse primers and probe for ribosomal 18S (all 50 nM; Applied Biosystems). Ribosomal 18S was used as a housekeeping gene. 1 ml of cDNA was added for each PCR. Internal control (liver cDNA) and negative control (water in place of cDNA) samples were included in each PCR run along with the RT negative and RT H2O control samples described above. All samples were analysed in triplicate using the 22DDCt method. PCRs were run on an ABI 7900 Sequence Detection System (Perkin-Elmer Applied Biosystems, USA). NOD1 and NOD2 primers and probes were designed using Primer Express software (Applied Biosystems). Primers and probes were validated and linearity of response was confirmed by serial dilution of cDNA. Primer and probe sequences and within assay variation are detailed in Table II. Immunohistochemistry Immunohistochemical localization of NOD1 and NOD2 was performed on endometrial (n 16 in total; n 4 proliferative & mid secretory; n 3 early & late secretory; n 2 menstrual) and decidual (n 4) sections using standard protocols. In brief, tissue sections were dewaxed in xylene and rehydrated in descending grades of alcohol. Sections were microwaved for 15 min in antigen unmasking solution (Vector) and then non-specific endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Sigma-Aldrich). Sections then underwent avidin, biotin (Avidin biotin blocking kit, Vector) and protein blocks (DakoCytomation protein block, Dako), each for 10 min at room temperature. Sections were incubated overnight at 48C with either rabbit-anti NOD1 (1:200; Imgenex) or rabbit anti-NOD2 (1:50; H-300, Santa Cruz) antibody diluted in Dako REAL antibody diluent (Dako). In negative control sections, the primary antibody was substituted with antibody diluent alone. A first trimester trophoblast section was included as a positive control for NOD2 immunostaining (Costello et al., 2007). Sections were subsequently incubated with biotinylated goat anti-rabbit Ig and were then subjected to an avidin biotin peroxidase detection system (both for 30 min at RT; Vectastain Elite ABC, Vector). Positive staining was detected using the peroxidase substrate, diaminobenzidine (ImmPACT DAB; Vector). Sections were counterstained with Harris haematoxylin, dehydrated in ascending grades of ethanol and mounted from xylene in Pertex. Cell culture Endometrial epithelial cells Telomerase-immortalized endometrial epithelial cells (EEpCs) (Hombach-Klonisch et al., 2005) were used as an in vitro model of EEpC. These cells were derived from a normal proliferative phase endometrial biopsy and expressed functional ERa and PR (Hombach-Klonisch et al., 2005). Cells were seeded in six-well culture plates at a density of 3 105/well. Cells were serum starved overnight prior to treatment with either: (i) the NOD1 ligand, D-g-Glu-mesodiaminopimelic acid (iE-DAP, 0.1 10 mg/ml, n 5; Invivogen); (ii) the NOD2 ligand, muramyl dipeptide (MDP, 0.1 10 mg/ml, n 4; Invivogen); or (iii) IL-1b (0.1 10 ng/ml, n 3; Peprotech); for 24 h. Cells were then harvested for RNA extraction and subsequent gene expression analysis by quantitative RT PCR (as detailed above). N numbers refer to separate experiments. Endometrial stromal cells Primary endometrial stromal cells (ESCs) were isolated from endometrial biopsies (n 7) and grown in culture as described previously (Kane et al., 2008). Cells were either: (i) seeded at 3 105/well in six-well culture plates, serum starved overnight and then treated with iE-DAP (0.1 10 mg/ml) for 24 h (n 3); or (ii) seeded at 2.4 105/well in six-well culture plates and decidualized in vitro by treatment with medroxyprogesterone acetate (MPA) (1026 M), estradiol (1028 M) and 8-bromo-cAMP (0.1 mg/ml) for 0, 24, 48, 72, 96 and 120 h (n 4). Decidualization was confirmed by measurement of IGFBP1 mRNA expression. Cells were harvested for RNA extraction and gene expression analysis by quantitative RT PCR (as detailed above). N numbers refer to separate experiments carried out using ESC derived from different endometrial biopsies. Statistics Data were logarithmically transformed prior to statistical analysis. Significant difference was determined by one-way ANOVA and Tukeys post hoc analysis. Results NOD1 and NOD2 mRNA is differentially expressed in human endometrium and first trimester decidua NOD1 NOD1 mRNA expression did not vary significantly in endometrium during the menstrual cycle or in first trimester decidua (Fig. 1a). NOD1 mRNA expression did not show any correlation with circulating serum estradiol (R2 0.0002; data not shown) or progesterone concentrations (R2 0.0275; data not shown). Figure 1 Differential mRNA expression of NOD1 and NOD2 in endometrium across the menstrual cycle and in first trimester decidua. P, proliferative; ES, early secretory; MS, mid-secretory; LS, late secretory; D, decidua (n 4 biopsies in each group). (a) NOD1. NOD1 mRNA expression does not vary significantly over the menstrual cycle or in first trimester decidua. (b) NOD2. NOD2 mRNA expression peaks in the late secretory of the menstrual cycle. abcd: P , 0.05. (c) NOD2 mRNA expression in biopsies collected during the secretory phase of the menstrual cycle shows a negative correlation with serum progesterone concentrations (R2 0.8746). NOD2 NOD2 mRNA expression was maximal in the late secretory phase of the menstrual cycle when levels were significantly higher than those in endometrium from all other cycle phases and in first trimester decidua (Fig. 1b; P , 0.05). NOD2 mRNA expression in biopsies collected during the secretory phase of the menstrual cycle showed a negative correlation with serum progesterone concentrations (R2 0.8746; Fig. 1c). There was no correlation between NOD2 mRNA expression and circulating estradiol concentrations (R2 0.0977; data not shown). NOD1 and NOD2 proteins are expressed in human endometrium and first trimester decidua NOD1 NOD1 was expressed in the glandular epithelium of endometrium from all menstrual cycle phases (Fig. 2a c). Additional immunostaining was present in some endothelial and stromal cells, particularly in stromal cells in the functional layer. There were no obvious changes in the pattern of localization at different phases of the menstrual cycle. NOD1 was expressed in the decidualized stroma and glandular epithelium of first trimester decidua (Fig. 2d). NOD2 NOD2 showed a similar localization profile to NOD1 and was localized predominantly to the glandular epithelium of endometrium from all cycle stages with additional immunostaining in the endothelium and isolated cells in the stroma (Fig. 2e g). Localization patterns did not differ with stage of the menstrual cycle. NOD2 was expressed in decidualized stromal cells and the glandular epithelium in first trimester decidua (Fig. 2h). NOD1 and NOD2 are functional in human endometrial epithelial and stromal cells in vitro The presence of NOD1 mRNA in ESC and EEpC was confirmed by quantitative RT PCR. NOD2 mRNA was present in EEpC but undetectable in ESC using this method (data not shown). Treatment of ESC with the NOD1 ligand, iE-DAP, resulted in increased mRNA expression of the NFkB inhibitor, IkBa (P , 0.05; Fig. 3a). Interleukin (IL-8) mRNA expression was not affected by iE-DAP (data not shown). Treatment of EEpC with the NOD1 ligand, iE-DAP and the NOD2 ligand, MDP, resulted in increased mRNA expression of IkBa (DAP, P , 0.05; MDP, P , 0.01; Fig. 3b). IL-8 and tumour necrosing factor (TNFa) mRNA expression was also increased by treatment with MDP (IL-8, P , 0.01, Fig. 3c; TNFa, P , 0.001, Fig. 3d) but was unaffected by iE-DAP. NOD1 and NOD2 mRNA expression is differentially regulated in human EEpC NOD1 NOD1 mRNA expression in EEpC was unchanged in response to treatment with IL-1b (Fig. 4a). NOD2 NOD2 mRNA expression in EEpC was significantly increased by 5-fold in the presence of IL-1b (Fig. 4b; P , 0.01). NOD1 mRNA expression is increased in an in vitro model of decidualization NOD1 Expression of NOD1 mRNA was raised 5-fold in an in vitro model of decidualization over a 120 h time-course (Fig. 5: P , 0.05). Discussion To our knowledge this is the first report detailing the expression, function and regulation of NOD1 and NOD2 in human endometrium. We have shown that both PRRs are present in human endometrium and first trimester decidua and that NOD2 functions to increase expression of the inflammatory molecules, IkBa, IL-8 and TNFa, in EEpC in vitro while treatment of EEpC and ESC with a NOD1 ligand results in increased expression of IkBa. There are few studies detailing expression of NODs in the female reproductive tract although a recent study has shown these PRRs to be present in first trimester trophoblast where they are suggested to contribute to the innate immune response of the placental villi upon encounter with invading pathogens (Costello et al., 2007). Our data show that NOD1 is constitutively expressed in endometrium while NOD2 mRNA expression is increased during the late secretory phase of the menstrual cycle. Immunohistochemical localization of NOD1 and NOD2 indicated wide expression in endometrium, predominantly in the epithelium but also in stromal cells and the endothelium. This is a very similar localization profile to that previously reported for TLRs in endometrium (Fazeli et al., 2005; Aflatoonian et al., 2007). The functional significance of cycle-dependent expression of innate immune molecules in the endometrium is unclear but is likely to relate to the need for increased immune protection during pivotal reproductive events. The TLR family of PRRs are widely expressed in the reproductive tract and, although the published data are conflicting, they have been shown to have cycle-dependent expression in endometrium coinciding with either menstruation (Hirata et al., 2007) or the putative window of implantation (Aflatoonian et al., 2007). Our previous studies examining expression of innate immune effector molecules such as secretory leukocyte protease inhibitor, elafin and human b-defensins in endometrium have shown these to be expressed predominantly in the latter half of the menstrual cycle, again at the time of the implantation window or coincident with menstruation (King et al., 2000, 2003b, c; Fleming et al., 2003). Successful implantation is dependent on prevention of uterine infection while the breaching of the epithelial barrier at menstruation may increase endometrial susceptibility to infection. Increased surveillance for pathogens at these times, for example by NOD2, may ensure a robust innate immune response if required. In addition to a role in innate immune defence, the increased expression of NOD2 in the late secretory phase of the menstrual cycle suggests a potential role in the physiological inflammatory mechanisms that underlie menstruation. Inflammatory mediators including IL-8 and TNFa show increased expression in the endometrium around the time of menstruation and there is leukocyte infiltration into the tissue (Tabibzadeh et al., 1995; Critchley et al., 1999; Jones et al., 2004; Jabbour et al., 2006). Menstruation also involves Figure 4 Differential regulation of NOD1 and NOD2 mRNA expression in EEpC (n 3 separate experiments). (a) NOD1. NOD1 mRNA is unchanged by treatment with the proinflammatory cytokine, IL-1b (0.1 10 ng/ml), for 24 h. (b) NOD2. NOD2 mRNA is increased by the presence of IL-1b. abc: P , 0.01. EEpc, endometrial epithelial cells. Figure 5 NOD1 mRNA expression is increased in an in vitro model of decidualization. Cells were decidualized in the presence of 8-medroxyprogesterone acetate (1026 M), estradiol (1028 M) and 8-bromo-cAMP (0.1 mg/ml) for 0 120 h (n 4 separate experiments). NOD1 mRNA expression increases 5-fold over the 120 h timecourse. ad: P , 0.05; bce: P , 0.001. resolution of inflammation and tissue remodelling and repair (Jabbour et al., 2006). While NOD2 may contribute to the regulation of inflammatory events at menstruation, a potential ligand responsible for NOD2 activation under these circumstances remains to be identified. There are studies implicating endogenous ligands such as heat shock proteins (de Graaf et al., 2006) and fibronectin (Okamura et al., 2001) in the activation of TLRs. These endogenous ligands are suggested to be released due to tissue damage under conditions of sterile inflammation and may initiate a response that ultimately leads to resolution of inflammation (Zhang and Schluesener 2006). While these studies remain controversial (Tsan and Baochong 2007), it may be that endogenous ligands can similarly activate NOD2 at menstruation, which is an example of sterile inflammation in the endometrium. The current study also implicates a role for NOD1 and NOD2 signalling in early pregnancy. Our immunohistochemical studies show that NOD1 and NOD2 expression is maintained in the glandular epithelium of first trimester decidua but the site of expression also shifts to the decidualized stroma. In an in vitro model NOD1 mRNA increases as decidualization of primary ESC progresses. Decidualized stromal cells have also been shown to express TLR2 and TLR4 (Krikun et al., 2007). As mentioned above, the endometrial epithelium is a key barrier to uterine infection and the presence of NOD1, NOD2 and other innate immune molecules in decidualized stromal cells may reflect an increased role in innate immune protection in response to the glandular atrophy that occurs in early pregnancy. Previous studies have shown that NOD1 and NOD2 function to increase expression of innate immune molecules at mucosal surfaces throughout the body. The natural antimicrobial molecule, betadefensin 2, was up-regulated by NOD ligands in epithelial cell lines representing several mucosal surfaces including those of the lung, cervix and breast (Uehara and Takada 2007; Uehara et al., 2007). Molecules that modulate the inflammatory response or have chemotactic activity such as IL-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) (Uehara and Takada 2007; Uehara et al., 2007) were up-regulated by NOD ligands in the HT29 and SW620 colonic epithelial cell lines and, in a separate study, were reported to increase in gingival fibroblasts (Uehara and Takada 2007). Our data show that, consistent with their role at other mucosal surfaces, NOD2 and possibly NOD1 are able to modulate the innate immune response in endometrial epithelial and/or stromal cells in response to their ligands. Activation of NOD1 and NOD2 increases expression of the NFkB inhibitor, IkBa. This suggests activation of the NFkB pathway as IkBa is an NFkB inducible gene. Previous studies have indicated that both NODs initiate the innate immune response via NFkB activation (Inohara et al., 2001; Ogura et al., 2001). In addition, our in vitro culture studies have shown that stimulation of EEpC with the NOD2 ligand, MDP, resulted in increased mRNA expression of the chemokine, IL-8 and the proinflammatory cytokine, TNFa. IL-8 promotes neutrophil recruitment and activation (Yoshimura et al., 1987), angiogenesis (Koch et al., 1992) and mitogenesis (Tuschil et al., 1992; Yue et al., 1994) while TNFa modulates the inflammatory response (Old 1985) and is involved in monocyte chemotaxis (Ming et al., 1987) and angiogenesis (Frater-Schroder et al., 1987). Increased production of these molecules during infection or menstruation is likely to enhance the innate immune response and promote tissue remodelling and regeneration as discussed above. We did not find any effect of the NOD1 ligand on IL-8 or TNFa mRNA expression in either EEpCs or ESCs. All in vitro treatments of cells with NOD ligands were performed for 24 h and it may be that a wider timecourse would have revealed a more convincing response to the NOD1 ligand. Further studies are required to confirm a functional role of NOD1 in endometrium. We have shown a negative correlation between circulating progesterone concentrations and NOD2 mRNA expression in endometrial biopsies, which is consistent with the up-regulation of NOD2 in the late secretory phase of the menstrual cycle. The late secretory phase is characterized by progesterone withdrawal and this may be directly responsible for the increase in NOD2 expression although this requires further investigation. Alternatively, the increased expression of inflammatory mediators in the late secretory phase (a consequence of progesterone withdrawal) may be involved in the regulation of NOD2 expression. We investigated the effects of the proinflammatory cytokine, IL-1b, on NOD expression in in vitro cell culture studies and our data suggest that NOD2 is upregulated in the endometrial epithelium by IL-1b while NOD1 is unaffected. NOD2 has previously been reported to be responsive to stimulation with inflammatory molecules in other systems and the NOD2 promoter region contains two kB sites indicating regulation by NFkB (Rosenstiel et al., 2003). NOD2 expression is increased by IL-1b in HUVEC cells (Oh et al., 2005), by TNFa, IL-1b and IL-6 in RAW264.7 macrophages (Takahashi et al., 2006) and by TNFa in several epithelial cell lines and primary colonic epithelial cells (Rosenstiel et al., 2003). In contrast to our data presented herein, NOD1 has also been reported to increase in response to treatment of RAW264.7 macrophages with TNFa, IL-1b and IL-6 (Takahashi et al., 2006) indicating that NOD1 regulation may be cell type specific. The increased expression of NOD2 mRNA in response to inflammatory mediators may be one mechanism by which NOD2 mRNA is raised in endometrium during the late secretory phase, a time when endometrial expression of inflammatory molecules such as IL-1, IL-8 and COX-2 increases (Critchley et al., 1999; Jabbour et al., 2006). In summary, NOD1 and NOD2 are differentially expressed and regulated in human endometrium and are likely involved in the innate immune response of this mucosal surface. NOD2 may also have a role in the physiological inflammatory events that are associated with menstruation. Aberrant expression or downstream signalling of NOD1 and/or NOD2 may increase susceptibility to uterine infection resulting in pathophysiological conditions such as predisposition to infertility and miscarriage, as well as aberrations in menstrual bleeding. Authors role A.E.K.: laboratory work, data analysis and manuscript preparation; A.W.H., H.O.D.C.: data analysis and manuscript preparation; S.H.K., J.I.M.: manuscript preparation. Acknowledgements We thank Catherine Murray and Sharon MacPherson in Edinburgh for recruitment of patients and collection of biopsies and Dr Vikki Abrahams (Yale University) for helpful discussions regarding NOD2 immunohistochemistry. This work was supported by a personal research fellowship from the Caledonian Research Foundation (Scotland) to A.K. and in part from funds from the Barbour Watson Foundation, Tenovus Scotland and MRC Programme Grant G0500047.


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Anne E. King, Andrew W. Horne, Sabine Hombach-Klonisch, J. I. Mason, Hilary O.D. Critchley. Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation, Molecular Human Reproduction, 2009, 311-319, DOI: 10.1093/molehr/gap020