Exploring the contribution of mammary-derived serotonin on liver and pancreas metabolism during lactation.

During lactation, the murine mammary gland is responsible for a significant increase in circulating serotonin. However, the role of mammary-derived serotonin in energy homeostasis during lactation is unclear. To investigate this, we utilized C57/BL6J mice with a lactation and mammary-specific deletion of the gene coding for the rate-limiting enzyme in serotonin synthesis (TPH1, Wap-Cre x TPH1FL/FL) to understand the metabolic contributions of mammary-derived serotonin during lactation. Circulating serotonin was reduced by approximately 50% throughout lactation in Wap-Cre x TPH1FL/FL mice compared to wild-type mice (TPH1FL/FL), with mammary gland and liver serotonin content reduced on L21. The Wap-Cre x TPH1FL/FL mice had less serotonin and insulin immunostaining in the pancreatic islets on L21, resulting in reduced circulating insulin but no changes in glucose. The mammary glands of Wap-Cre x TPH1FL/FL mice had larger mammary alveolar areas, with fewer and smaller intra-lobular adipocytes, and increased expression of milk protein genes (e.g., WAP, CSN2, LALBA) compared to TPH1FL/FL mice. No changes in feed intake, body composition, or estimated milk yield were observed between groups. Taken together, mammary-derived serotonin appears to contribute to the pancreas-mammary cross-talk during lactation with potential implications in the regulation of insulin homeostasis.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches - mainly antibiotic treatments - are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

Gross anatomy of vascular supply and drainage of mammary fat pads in mice models.

This work extensively studied the vasculature of mice mammary fat pads (BALB/c and C57BL/6) with special reference to haematogenous drainage routes. Mammary fat pads were five pairs (first cervical, second and third thoracic, fourth abdominal and fifth inguinal), bilaterally symmetrical, extending laterally and continuously with the subcutaneous fascia. The superficial cervical artery and vein primarily accomplished the blood vasculature of the first mammary fat pad, while the lateral thoracic and external thoracic arteries and veins supplied the second and third mammary fat pads. The superficial cervical vein (found parallel to the superficial cervical artery) drained into the external jugular vein. The lateral thoracic artery and external thoracic artery branched almost at the same level as the axillary artery (branch of subclavian artery), the latter being more medial in position. However, in some specimens, the branching of both arteries appeared to be at the same level, and their origins were indistinguishable. The lateral thoracic vein that was parallel to the lateral thoracic artery drained to the axillary vein close to the drainage of the external thoracic vein. The lateral thoracic, superficial caudal epigastric, iliolumbar and external thoracic arteries and veins vascularized the fourth mammary fat pad and displayed anastomosis among themselves. The iliolumbar vein (found parallel to the iliolumbar artery) drained into the inferior vena cava. The superficial caudal epigastric vein (found parallel to the superficial caudal epigastric artery (SCaEA)) drained into the femoral vein. Unlike humans, the internal thoracic artery and vein did not participate in the vasculature of mammary fat pads. The SCaEA and vein supplied blood and drained the fifth mammary fat pad. The anatomical continuity of the fourth and fifth mammary fat pads provided common drainage for both mammary fat pads. The BALB/c and C57BL/6 mice strains studied did not differ in topography and size of mammary fat pads. The vascular supply and drainage of the mammary fat pads also did not differ in the strains studied. Only minor variations could be noted in the small veins draining into the lateral thoracic vein. Lateral tributaries seen in the terminal end of the lateral thoracic vein were absent in the C57BL/6 mice.

Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling.

Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

Role of LsrR in the regulation of biofilm formation in mammary pathogenic Escherichia coli.

BACKGROUND: Mammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported. RESULTS: In this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner. CONCLUSIONS: These results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC.

MicroRNA-2285f regulates milk fat metabolism by targeting MAP2K2 in bovine mammary epithelial cells.

In this study, Holstein dairy cows raised in Ningxia were selected as the research object. Mammary epithelial cells (BMECs) were extracted from the milk of eight Holstein cows with significantly different milk fat expression rates and transcribed for sequencing. Bioinformatics analysis was used to analyse the correlation of fat milk percentage, and the critical miR-2285f regulating milk fat was screened out. The target gene binding sites were predicted, and 293T cells and mammary epithelial cells were used as miRNA and target gene models for functional verification in vitro. The tissue difference of miR-2285f Holstein cows was quantitatively analysed by transfecting miR-2285f mimic and inhibitor. Assay (dual luciferase reporter gene assay) and quantitative real-time PCR (quantitative real-time PCR, qRT-PCR), triglyceride (TAG) detection, oil red O detection of lipid droplets, Western Blot assay, Edu and Flow cytometry, The molecular regulatory effects of miR-2285f and target gene MAP2K2 on milk fat metabolism of Holstein dairy cows were studied. The wild-type vector and mutant vector of map2k2-3'utr were constructed, and double luciferase reporting experiments were conducted to verify that MAP2K2 was one of the target genes of miR-2285f. According to qRT-PCR and Western Blot analysis, miR-2285f mainly regulates the expression of MAP2K2 protein in BMECs at the translation level. Bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2. Bta-miR-2285f can promote triglyceride (TAG) and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2. Bta-miR-2285f can regulate protein levels of fat milk marker gene PPARG by targeting MAP2K2. In conclusion, miR-2285f can target the expression of the MAP2K2 gene, promote the proliferation of dairy mammary epithelial cells, inhibit cell apoptosis and regulate the milk fat metabolism in dairy mammary epithelial cells. The results of this study revealed the function of miR-2285f in regulating the differential expression of fat milk in Holstein dairy cows at the cellular level. They provided a theoretical and experimental basis for analysing the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.

Ultrasonographic, endoscopic and radiographic examinations of the dromedary mammary glands and teats.

The aim of the present study was to examine the mammary gland of dromedary camels using ultrasonography, endoscopy and radiography. These techniques are easy to perform in the field and feasible to diagnose pathological conditions of the mammary gland. Udders of 49 slaughtered and 26 adult dromedary camels submitted for necropsy were used for the examinations. Additionally, 11 lactating female dromedary camels were selected for the ultrasonographic udder examination. The transition from the milk ducts into the udder cistern, the teat cistern and the teat canals were examined in individual udders. Teat cistern length, teat end width, teat wall thickness, teat cistern width and middle cistern wall thickness were measured using ultrasonography. The measurements resulted in mean values of the teat cistern length of 37.3 mm, the teat end width of 2.0 mm, the teat wall thickness of 4.4 mm, the teat cistern width of 8.2 mm and the cistern wall thickness of 3.5 mm. The teat wall was differentiated into three layers, a hyperechoic outer layer, a hypoechoic middle layer and a hyperechoic inner layer. The mid cistern wall was hyperechoic. Endoscopic examination is an easy to perform and practicable method for examining the inner structures of the teats of dead animals; however, the feasibility has not been shown in lactating animals yet. Ring-like folds were present in the teat cistern, which protruded horizontally into the lumen. It was also possible to visualize the branchlike transition of the teat cistern into the larger milk ducts. Radiographic examination using barium sulfate contrast medium showed that the teat cistern ends in a network of initially wide but branching and narrowing milk ducts. The two teat canals and cisterns are completely independent of each other and there is no communication between the glandular tissue of the two canals and cisterns.

Retrospective evaluation of udder recovery of cows with subclinical mastitis following treatment with acoustic pulse technology (APT) on commercial dairy farms and its economic impact.

Retrospective evaluation of udder recovery following treatment of the inflamed quarter with acoustic pulse technology (APT) of cows with subclinical mastitis was done on 4 Israeli commercial dairy farms. Here, we evaluated the APT treatment as a tool to manage subclinical mastitis and its economic consequences in commercial farms. Recovery of the infected glands following APT treatment was compared to the customary no-treatment (NT) for cows with subclinical mastitis. Over 2 years, 467 cows with subclinical mastitis were identified. Subclinical mastitis was defined by elevated somatic cell count (SCC; >1 × 106 cells/mL) in the monthly test-day milk sample; 222 cows were treated with APT and 245 cows were not treated and served as control. Differences between treatment groups in culling, milk quality, milk yield and bacterial elimination were analyzed. After treatment, cure from bacteria was calculated only for cows with pre-isolated bacteria. The percentage of sampled cows determined as cured (no bacterial finding) in the NT group was 32.7% (35/107) (30.9% Gram negative; 32.4% Gram positive) and in the APT-treated group, 83.9% (42/55) (89.4% Gram negative; 80.6% Gram positive). Culling rate due to mastitis was significantly lower (>90%) in the APT-treated vs. NT group. Recovery was 66.0% in the APT group compared to 11.5% in the NT group at 90 d post-treatment. Average milk volume per cow in the APT-treated group was 16.1% higher compared to NT cows. Based on the study, savings incurred by using APT to treat only subclinical cows per 100-cow herd can total $15,106/y, or $309 per treated subclinically infected cow.

To probe outbreak, BSL-3 labs plan to infect cows with flu virus.

Novel effort comes as study finds key receptor for avian flu virus in udders, where the virus flourishes.

LYVE-1-expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth.

Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peritumoral stroma, their functions within these regions are not well understood. Using a genetic mouse model of LYVE-1+ macrophage depletion, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that depletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single-cell RNA sequencing of macrophages isolated from these tumors reveals that depletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages toward a proinflammatory phenotype, as well as an increase in CD8+ T-cell infiltration. Together, these findings indicate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment. SIGNIFICANCE: We have identified a macrophage subset in mouse mammary tumors associated with tumor structural components. When this macrophage subset is absent in tumors, we report a delay in tumor growth and an increase in antitumor immune cells. Understanding the functions of distinct macrophage subsets may allow for improved therapeutic strategies for patients with breast cancer.

In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

Regulatory roles of dopamine D2 receptor in milk protein production and apoptosis in mammary epithelial cells.

Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs.

ABCD4 is associated with mammary gland development in mammals.

BACKGROUND: Mammary gland development is a critical process in mammals, crucial for their reproductive success and offspring nourishment. However, the functional roles of key candidate genes associated with teat number, including ABCD4, VRTN, PROX2, and DLST, in this developmental process remain elusive. To address this gap in knowledge, we conducted an in-depth investigation into the dynamic expression patterns, functional implications, and regulatory networks of these candidate genes during mouse mammary gland development. RESULTS: In this study, the spatial and temporal patterns of key genes were characterized in mammary gland development. Using time-series single-cell data, we uncovered differences in the expression of A bcd4, Vrtn, Prox2, and Dlst in cell population of the mammary gland during embryonic and adult stages, while Vrtn was not detected in any cells. We found that only overexpression and knockdown of Abcd4 could inhibit proliferation and promote apoptosis of HC11 mammary epithelial cells, whereas Prox2 and Dlst had no significant effect on these cells. Using RNA-seq and qPCR, further analysis revealed that Abcd4 can induce widespread changes in the expression levels of genes involved in mammary gland development, such as Igfbp3, Ccl5, Tlr2, and Prlr, which were primarily associated with the MAPK, JAK-STAT, and PI3K-AKT pathways by functional enrichment. CONCLUSIONS: These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Host response during unresolved urinary tract infection alters female mammary tissue homeostasis through collagen deposition and TIMP1.

Exposure to pathogens throughout a lifetime influences immunity and organ function. Here, we explore how the systemic host-response to bacterial urinary tract infection (UTI) induces tissue-specific alterations to the mammary gland. Utilizing a combination of histological tissue analysis, single cell transcriptomics, and flow cytometry, we identify that mammary tissue from UTI-bearing mice displays collagen deposition, enlarged ductal structures, ductal hyperplasia with atypical epithelial transcriptomes and altered immune composition. Bacterial cells are absent in the mammary tissue and blood of UTI-bearing mice, therefore, alterations to the distal mammary tissue are mediated by the systemic host response to local infection. Furthermore, broad spectrum antibiotic treatment resolves the infection and restores mammary cellular and tissue homeostasis. Systemically, unresolved UTI correlates with increased plasma levels of the metalloproteinase inhibitor, TIMP1, which controls extracellular matrix remodeling and neutrophil function. Treatment of nulliparous and post-lactation UTI-bearing female mice with a TIMP1 neutralizing antibody, restores mammary tissue normal homeostasis, thus providing evidence for a link between the systemic host response during UTI and mammary gland alterations.