Adipocyte-induced transdifferentiation of osteoblasts and its potential role in age-related bone loss

Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.

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Age-related accumulation of advanced oxidation protein products promotes osteoclastogenesis through disruption of redox homeostasis

Cell Death and Disease ◽

10.1038/s41419-021-04441-w ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Jingshen Zhuang ◽

Xuebing Chen ◽

Guixing Cai ◽

Dizheng Wu ◽

Chen Tu ◽

...

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Nadph Oxidase ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Advanced Oxidation ◽

Oxidase Inhibitor ◽

Nuclear Factor ◽

Advanced Oxidation Protein Products ◽

Age Related

AbstractEnhanced osteoclastogenesis is one of the major causes of age-related bone loss. Aging is accompanied by accumulation of advanced oxidation protein products (AOPPs). However, whether AOPPs accumulation contributing to the osteoclastogenesis with aging remains unclear. Here, we showed that AOPPs accumulation was associated with the enhanced osteoclastogenesis and deterioration of bone microstructure in aged mice. In vitro, AOPPs directly induced osteoclastogenesis by interaction with receptor activator of nuclear factor κ B (RANK) and the receptor for advanced glycation end products (RAGE) in the primary bone marrow monocytes. Bindings of AOPPs to RANK and RAGE were able to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, trigger generation of reactive oxygen species, then induce phosphorylation of mitogen-activated protein kinases and c-fos, upregulation of the nuclear factor of activated T cell c1, eventually induce bone marrow monocytes to differentiate into mature osteoclasts. Chronic exposure to AOPPs enhanced osteoclastogenesis and bone loss in mice, which could be alleviated by NADPH oxidase inhibitor apocynin. Local injection of AOPPs into subperiosteal area induced bone resorption at the site of administration, which was similar to the effect of RANK ligand. Together, these results suggested that AOPPs could serve as a novel regulator of osteoclastogenesis and AOPPs accumulation might play an important role in the development of age-related bone loss.

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AGE-ASSOCIATED INCREASE IN KYNURENINE INHIBITS AUTOPHAGY AND PROMOTES SENESCENCE IN BONE MARROW STEM CELLS

Innovation in Aging ◽

10.1093/geroni/igz038.3469 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S956-S956

Author(s):

Dmitry Kondrikov ◽

Ahmed Elmansi ◽

Xing-ming Shi ◽

Sadanand Fulzele ◽

Meghan mcGee-Lawrence ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Lines ◽

Free Radical Oxidation ◽

Autophagic Flux ◽

Galactosidase Activity ◽

Activity Increase ◽

Age Related ◽

Old Mice

Abstract Aging is characterized by progressive decline of tissue functionality and age-related accumulation of cellular and molecular damage leading to multiple pathological conditions including osteoporosis and increased fracture rates. Bone marrow mesenchymal stem cells (BMSCs) play an essential role in bone development and regeneration with their ability to undergo differentiation into osteogenic, chondrogenic, myogenic, and adipogenic cell lines cell lines. Proliferation rate of MSC is declined with ages leading to misbalance between bone resorption and osteogenesis. A recently identified age-related change in bone and bone marrow is an accumulation of tryptophan metabolite, kynurenine (KYN), catalyzed by indoleamine-2,3-dioxygenase (IDO) or free-radical oxidation. We previously reported that KYN suppresses autophagy in BMSC. We now investigated the effect of KYN on BMSC cellular function. In vitro treatment of murine BMSC isolated from 18 month old mice with kynurenine disrupted autophagy suppressing autophagic flux. KYN treatment also induces senescence in BMSC marked by increase in SA-beta-galactosidase activity as well as, increased expression of senescence marker p21. Inhibition of Aryl Hydrocarbon Receptor (AhR) by AhR inhibitors significantly reduced β-galactosidase activity increase and blocked p21 expression elevation suggesting that KYN induces senescence in BMSC through the AhR pathway. Interestingly, KYN treatment failed to up-regulate beta-gal activity in BMSC isolated from 6 month-old mice suggesting that KYN induction of senescence maybe potentiated with aging. Together those data support the idea that KYN shifts the homeostatic balance of BMSC during prolonged stress or in aging through downregulating survival autophagic pathway in favor of driving BMSCs to senescence.

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Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss

International Journal of Molecular Sciences ◽

10.3390/ijms21082745 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2745

Author(s):

Yukihiro Kohara ◽

Ryuma Haraguchi ◽

Riko Kitazawa ◽

Yuuki Imai ◽

Sohei Kitazawa

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Signaling Pathway ◽

Early Stage ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Age Related ◽

Hh Signaling

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0–48 h (early stage of osteoclast differentiation) or 48–96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

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Characterization of Hematopoietic Stem Cell Frequency and Function In Unexplained Anemia of the Elderly

Blood ◽

10.1182/blood.v116.21.2047.2047 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2047-2047

Author(s):

Wendy Pang ◽

Elizabeth Price ◽

Irving L. Weissman ◽

Stanley L. Schrier

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

In Vitro Culture ◽

Progenitor Cells ◽

The Elderly ◽

Elderly Subjects ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

And Function

Abstract Abstract 2047 Anemia is both a highly prevalent and clinically important condition that causes significant morbidity and mortality in the elderly population. While anemia in the elderly can be attributed to a number of causes, approximately 30% of elderly subjects with anemia have no overt etiology and fall under the category of unexplained anemia of the elderly (UA). There is increasing evidence to suggest that changes in the frequency and/or function of hematopoietic stem and progenitor cells may contribute to the onset and pathophysiology of age-associated hematological conditions, such as UA. Hematopoietic stem cells (HSC) reside at the top of the hematopoietic hierarchy and can differentiate, via increasingly committed downstream progenitors, into all the mature cells of the hematopoietic system. Human myelo-erythroid development proceeds through a set of oligopotent progenitors: HSC give rise to multipotent progenitors (MPP), which give rise to common myeloid progenitors (CMP), which in turn give rise to granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP). We use flow cytometry and in vitro culture of sorted human HSC (Lin-CD34+CD38-CD90+CD45RA-), MPP (Lin-CD34+CD38-CD90-CD45RA-), CMP (Lin-CD34+CD38+CD123+CD45RA-), GMP (Lin-CD34+CD38+CD123+CD45RA+), and MEP (Lin-CD34+CD38+CD123-CD45RA-) from hematologically normal young (23 samples; age 20–35) and elderly (11 samples; age 65+) and UA (5 samples; age 65+) bone marrow samples in order to characterize the changes in the distribution and function of hematopoietic stem and progenitor populations during the aging process and, in particular, in the development of UA. We found that UA patients contain higher frequencies of HSC compared to both elderly normal (1.5-fold; p<0.03) and young normal samples (2.8-fold; p<10-5). We also found increased frequencies of MPP from UA patients compared to MPP from elderly normal (2.6-fold; p<0.002) and young normal samples (5.8-fold; p<0.04). While we observed similar frequencies of CMP among the three groups, we found a notable trend suggesting decreased frequencies of GMP and corresponding increased frequencies of MEP in UA patients. Functionally, HSC from the three groups exhibit statistically insignificant differences in the efficiency of colony formation under the myeloid differentiation-promoting methylcellulose-based in vitro culture conditions; however, on average, HSC from elderly bone marrow samples, regardless of the presence or absence of anemia, tend to form fewer colonies in methylcellulose. Interestingly, HSC from UA patients produce more granulocyte-monocyte (CFU-GM) colonies and fewer erythroid (CFU-E and BFU-E) colonies, compared to HSC from normal samples (p<0.001). Similarly, CMP from UA patients, compared to normal CMP, yield skewed distributions of myeloid-erythroid colonies when plated in methylcellulose, significantly favoring production of CFU-GM colonies over CFU-E and BFU-E colonies (p<0.003). Additionally, MEP from UA patients form both CFU-E and BFU-E colonies in methylcellulose albeit at a significantly lower efficiency than MEP from normal bone marrow samples (p<0.01). This is the first study to examine the changes in hematopoietic stem and progenitor populations in UA patients. The changes in the distribution of hematopoietic stem and progenitor cells in UA patients indicate that the HSC and MPP populations, and possibly also the MEP population, expand in the context of anemia, potentially in response to homeostatic feedback mechanisms. Nevertheless, these expanded populations are functionally impaired in their ability to differentiate towards the erythroid lineage. Our data suggest that there are intrinsic defects in the HSC population of UA patients that lead to poor erythroid differentiation, which can be readily observed even in the earliest committed myelo-erythroid progenitors. We have generated gene expression profiling data from these purified hematopoietic stem and progenitor populations from UA patients to try to identify biological pathways and markers relevant to disease pathogenesis and potential therapeutic targets. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Schrier:Celgene: Research Funding.

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miR-542-3p Attenuates Bone Loss and Marrow Adiposity Following Methotrexate Treatment by Targeting sFRP-1 and Smurf2

International Journal of Molecular Sciences ◽

10.3390/ijms222010988 ◽

2021 ◽

Vol 22 (20) ◽

pp. 10988

Author(s):

Ya-Li Zhang ◽

Liang Liu ◽

Yu-Wen Su ◽

Cory J. Xian

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Target Prediction ◽

In Vitro Models ◽

Bone Homeostasis ◽

Marrow Fat ◽

Childhood Malignancies ◽

Marrow Adiposity ◽

Underlying Mechanisms

Intensive methotrexate (MTX) treatment for childhood malignancies decreases osteogenesis but increases adipogenesis from the bone marrow stromal cells (BMSCs), resulting in bone loss and bone marrow adiposity. However, the underlying mechanisms are unclear. While microRNAs (miRNAs) have emerged as bone homeostasis regulators and miR-542-3p was recently shown to regulate osteogenesis in a bone loss context, the role of miR-542-3p in regulating osteogenesis and adipogenesis balance is not clear. Herein, in a rat MTX treatment-induced bone loss model, miR-542-3p was found significantly downregulated during the period of bone loss and marrow adiposity. Following target prediction, network construction, and functional annotation/ enrichment analyses, luciferase assays confirmed sFRP-1 and Smurf2 as the direct targets of miR-542-3p. miRNA-542-3p overexpression suppressed sFRP-1 and Smurf2 expression post-transcriptionally. Using in vitro models, miR-542-3p treatment stimulated osteogenesis but attenuated adipogenesis following MTX treatment. Subsequent signalling analyses revealed that miR-542-3p influences Wnt/β-catenin and TGF-β signalling pathways in osteoblastic cells. Our findings suggest that MTX treatment-induced bone loss and marrow adiposity could be molecularly linked to miR-542-3p pathways. Our results also indicate that miR-542-3p might be a therapeutic target for preserving bone and attenuating marrow fat formation during/after MTX chemotherapy.

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IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽

10.1182/blood-2020-137026 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Yang Mei ◽

Yijie Liu ◽

Xu Han ◽

Jing Yang ◽

Peng Ji

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Inflammatory Cytokines ◽

Conflicts Of Interest ◽

Double Knockout ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Cell Counts ◽

Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

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Mixed lineage kinase domain-like pseudokinase mediated necroptosis aggravates periodontitis progression

10.21203/rs.3.rs-468306/v1 ◽

2021 ◽

Author(s):

Yanan Yang ◽

Lingxia Wang ◽

Haibing Zhang ◽

Lijun Luo

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Porphyromonas Gingivalis ◽

Alveolar Bone ◽

Alveolar Bone Loss ◽

Mrna Levels ◽

Genetic Ablation ◽

Osteoclast Activation

Abstract Necroptosis is a form of cell death that is reportedly involved in the pathogenesis of periodontitis. However, the role of Mlkl-involved necroptosis remains unclear. Herein, we aim to explore the role of MLKL-mediated necroptosis in periodontitis in vitro and in vivo. Expression of RIPK3, MLKL, and phosphorylated MLKL is observed in gingival tissues obtained from healthy subjects or patients with periodontitis. Viability of Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells was detected. In wild type or Mlkl deficiency mice with ligature-induced periodontitis, alveolar bone loss and osteoclast activation were assessed. mRNA levels of inflammatory cytokines in bone marrow-derived macrophages were tested by qRT-PCR. Increased expression of RIPK3, MLKL, and phosphorylated MLKL is observed in gingival tissues obtained from patients with periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells developed necroptosis after caspase inhibition and negatively regulated the NF-κB signaling pathway. In mice with ligature-induced periodontitis, Mlkl deficiency reduced alveolar bone loss and weakened osteoclast activation. Furthermore, genetic ablation of Mlkl in LPS-Pg-treated bone marrow-derived macrophages increased the mRNA levels of tumor necrosis factor-α, interleukin (Il)-1β, Il-6, cyclooxygenase 2, matrix metalloproteinase 9, and receptor activator of nuclear factor kappa-B ligand. Our data indicated that MLKL-mediated necroptosis aggravates the development of periodontitis in a Mlkl-deficient mouse. And this will provide a new sight for the understanding of etiology and therapies of periodontitis.

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miR-6315 Attenuates Methotrexate Treatment-Induced Decreased Osteogenesis and Increased Adipogenesis Potentially through Modulating TGF-β/Smad2 Signalling

Biomedicines ◽

10.3390/biomedicines9121926 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1926

Author(s):

Ya-Li Zhang ◽

Liang Liu ◽

Yu-Wen Su ◽

Cory J. Xian

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Bone Marrow Stromal Cells ◽

Molecular Mechanisms ◽

Adipogenic Differentiation ◽

Marrow Fat ◽

Childhood Malignancies ◽

Marrow Adiposity ◽

Underlying Mechanisms

Methotrexate (MTX) treatment for childhood malignancies has shown decreased osteogenesis and increased adipogenesis in bone marrow stromal cells (BMSCs), leading to bone loss and bone marrow adiposity, for which the molecular mechanisms are not fully understood. Currently, microRNAs (miRNAs) are emerging as vital mediators involved in bone/bone marrow fat homeostasis and our previous studies have demonstrated that miR-6315 was upregulated in bones of MTX-treated rats, which might be associated with bone/fat imbalance by directly targeting Smad2. However, the underlying mechanisms by which miR-6315 regulates osteogenic and adipogenic differentiation require more investigations. Herein, we further explored and elucidated the regulatory roles of miR-6315 in osteogenesis and adipogenesis using in vitro cell models. We found that miR-6315 promotes osteogenic differentiation and it alleviates MTX-induced increased adipogenesis. Furthermore, our results suggest that the involvement of miR-6315 in osteogenesis/adipogenesis regulation might be partially through modulating the TGF-β/Smad2 signalling pathway. Our findings indicated that miR-6315 may be important in regulating osteogenesis and adipogenesis and might be a therapeutic target for preventing/attenuating MTX treatment-associated bone loss and marrow adiposity.

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Quantification and Comprehensive Analysis of Mesenchymal Stromal Cells in Bone Marrow Samples from Sickle Cell Disease Patients with Osteonecrosis

Stem Cells International ◽

10.1155/2020/8841191 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Tiago O. Ribeiro ◽

Paula B. Daltro ◽

Gildasio Cerqueira Daltro ◽

Songeli M. Freire ◽

Roberto Meyer ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cell Number ◽

Cell Phenotype ◽

Cell Disease ◽

Age Related

The potential use of bone marrow mesenchymal stromal cells (BM-MSCs) for the treatment of osteonecrosis in sickle cell disease (SCD) patients is increasing. However, convenient BM-MSC quantification and functional property assays are critical factors for cell-based therapies yet to be optimized. This study was designed to quantify the MSC population in bone marrow (BM) samples from SCD patients with osteonecrosis (SCD group) and patients with osteoarticular complications not related to SCD (NS group), using flow cytometry for CD271+CD45-/low cell phenotype and CFU-F assay. We also compared expanded BM-MSC osteogenic differentiation, migration, and cytokine secretion potential between these groups. The mean total cell number, CFU-F count, and CD271+CD45-/low cells in BM mononuclear concentrate were significantly higher in SCD than in NS patients. A significant correlation between CD271+CD45-/low cell number and CFU-F counts was found in SCD ( r = 0.7483 ; p = 0.0070 ) and NS ( r = 0.7167 ; p = 0.0370 ) BM concentrates. An age-related quantitative reduction of CFU-F counts and CD271+CD45-/low cell number was noted. Furthermore, no significant differences in the morphology, replicative capacity, expression of surface markers, multidifferentiation potential, and secretion of cytokines were found in expanded BM-MSCs from SCD and NS groups after in vitro culturing. Collectively, this work provides important data for the suitable measurement and expansion of BM-MSC in support to advanced cell-based therapies for SCD patients with osteonecrosis.

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Immunological mechanisms to establish embryo tolerance in early bovine pregnancy

Reproduction Fertility and Development ◽

10.1071/rd10230 ◽

2011 ◽

Vol 23 (5) ◽

pp. 619 ◽

Cited By ~ 29

Author(s):

A. E. Groebner ◽

K. Schulke ◽

J. C. Schefold ◽

G. Fusch ◽

F. Sinowatz ◽

...

Keyword(s):

In Situ Hybridisation ◽

Immunohistochemical Analysis ◽

Kynurenine Pathway ◽

Endometrial Cells ◽

Maternal Rejection ◽

Immunological Mechanisms ◽

Bovine Pregnancy ◽

Coculture Model

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P < 0.001) more abundant in the endometrium of Day 18 pregnant v. non-pregnant animals. Tandem mass spectrometry revealed a decrease of endometrial l-tryptophan (P = 0.0008), but an increase of l-kynurenine concentration (P = 0.005) from Day 12 to Day 18, suggesting increasing IDO activity (P < 0.03). An in vitro coculture model of endometrial cells showed an induction of IDO expression following interferon-τ exposure primarily in stroma cells, which was confirmed by in situ hybridisation localising IDO mRNA mainly in deep stroma cells. Immunohistochemical analysis revealed fewer CD45-positive leucocytes in the zona basalis of pregnant animals. Elevated IDO activity may reduce the presence of leucocytes in the pregnant endometrium, providing a possible mechanism for protecting the semi-allogenic conceptus from maternal rejection.

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