Isoform-specific knockdown of long and intermediate prolactin receptors interferes with evolution of B-cell neoplasms.

Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell malignancies is unknown. We measure changes in B cells after knocking down the pro-proliferative, anti-apoptotic long isoform of the PRL receptor (LFPRLR) in vivo in systemic lupus erythematosus (SLE)- and B-cell lymphoma-prone mouse models, and the long plus intermediate isoforms (LF/IFPRLR) in human B-cell malignancies. To knockdown LF/IFPRLRs without suppressing expression of the counteractive short PRLR isoforms (SFPRLRs), we employ splice-modulating DNA oligomers. In SLE-prone mice, LFPRLR knockdown reduces numbers and proliferation of pathogenic B-cell subsets and lowers the risk of B-cell transformation by downregulating expression of activation-induced cytidine deaminase. LFPRLR knockdown in lymphoma-prone mice reduces B-cell numbers and their expression of BCL2 and TCL1. In overt human B-cell malignancies, LF/IFPRLR knockdown reduces B-cell viability and their MYC and BCL2 expression. Unlike normal B cells, human B-cell malignancies secrete autocrine PRL and often express no SFPRLRs. Neutralization of secreted PRL reduces the viability of B-cell malignancies. Knockdown of LF/IFPRLR reduces the growth of human B-cell malignancies in vitro and in vivo. Thus, LF/IFPRLR knockdown is a highly specific approach to block the evolution of B-cell neoplasms.

A cautionary tale: Alien prolactins may induce lesser, no, or opposite effects to homologous hormone!

Cost and availability have often dictated the use of heterologous/alien prolactins in experiments, particularly in vivo. The assumption has been that what is initiated in the target cell is representative of the homologous hormone since many heterologous mammalian prolactins bind to and activate rodent receptors. Here, we examined gene expression in mouse liver in response to a 7-day treatment with recombinant mouse prolactin (mRecPRL), recombinant ovine prolactin (oRecPRL) and pituitary extract ovine prolactin (oPitPRL). Having established mouse ribosomal protein S9 as the most stable reference gene in the liver in the absence and presence of prolactin treatment, we examined expression of the two most highly expressed prolactin receptors (PRLRs) and three members of the Cyp3a group of cytochrome P450 isoenzymes by qRTPCR. For short form (SF) 3 PRLR, mRecPRL doubled expression while for oRecPRL and oPitPRL expression was only 1.3-fold control. For the long form (LF) PRLR, changes were similar to those seen for SF 3 PRLR, such that the SF3:LF PRLR ratio remained the same. Expression of the Cyp3as was also dependent on the prolactin origin and, although mRecPRL always stimulated, the other PRLs caused varying results. Compared to control, Cyp3a16 was stimulated 12-fold by mRecPRL, 3-fold by oRecPRL, and 6-fold by oPitPRL. For Cyp3a41, mRecPRL was 3.7-fold control, oRecPRL was without effect, and oPitPRL was 2-fold control. Importantly, for Cyp3a44, mRecPRL stimulated 2-fold, whereas both oRecPRL and oPitPRL had an opposite, inhibitory effect, with expression at 0.5-fold control. We conclude that homologous hormone had the largest stimulatory effect on expression of all measured genes and that by contrast heterologous hormone showed reduced activity, no activity, or opposite activity, depending on the gene being analyzed. Thus, experimentation using alien heterologous PRL may lead to inaccurate conclusions.

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization.

BACKGROUND: Asymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known. METHODS: Three mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system. RESULTS: For macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor. CONCLUSION: Our results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF-tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7-day) showed prolactin-induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single-cell RNAseq, and analysis of LF-td-Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

Bittersweet: relevant amounts of the common sweet food additive, glycerol, accelerate the growth of PC3 human prostate cancer xenografts.

OBJECTIVE: In a study of potential prostate cancer therapeutics, glycerol was used to increase the density of one solution. Glycerol alone was therefore one of the controls. Tumors of human PC3 castrate-resistant prostate cancer cells were initiated in male nude mice and grown for 12 days. Mice were then sorted such that mean tumor weights were the same in each group, and osmotic minipumps delivering 0.25 µL/h of either saline or glycerol were then implanted subcutaneously. RESULTS: Contrary to our initial assumption that glycerol would be without effect, tumors grew more rapidly in the glycerol group such that tumors were twice the size of those in the saline group after 4 weeks. Given the dose delivered, analysis of the literature suggests this effect was not via the conversion of glycerol to glucose but possibly via a reduction in oxidative damage in the growing tumor. Our data demonstrate that amounts of glycerol that could reasonably be derived from the diet promote the growth of these tumors. Given the increasing use of glycerol in foods and beverages, we present these data to stimulate interest in an epidemiological study in the human population examining glycerol consumption and the aggressiveness of prostate cancer.

Prolactin Attenuates Neuroinflammation in LPS-Activated SIM-A9 Microglial Cells by Inhibiting NF-κB Pathways Via ERK1/2.

Prolactin (PRL) is a pleiotropic hormone with multiple functions in several tissues and organs, including the brain. PRL decreases lesion-induced microgliosis and modifies gene expression related to microglial functions in the hippocampus, thereby providing a possible mechanism through which it might participate in neuroimmune modulatory responses and prevent neuronal cell damage. However, the direct contribution of microglial cells to PRL-mediated neuroprotection is still unclear and no studies have yet documented whether PRL can directly activate cellular pathways in microglial cells. The aim of this study is to elucidate in vitro actions of PRL on the immortalized SIM-A9 microglia cell line in basal and LPS-stimulated conditions. PRL alone induced a time-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Pretreatment with PRL attenuated LPS (200 ng/ml) stimulated pro-inflammatory markers: nitric oxide (NO) levels, inducible nitric oxide synthase (iNOS), interleukins (IL)-6, -1ß and tumor necrosis factor (TNF-α) expression at 20 nM dosage. PRL suppressed LPS-induced nuclear factor (NF)-κappaB (NF-κB) p65 subunit phosphorylation and its upstream p-ERK1/2 activity. In conclusion, PRL exhibits anti-inflammatory effects in LPS-stimulated SIM-A9 microglia by downregulating pro-inflammatory mediators corresponding to suppression of LPS-activated ERK1/2 and NF-κB phosphorylation.

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation.

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 µm, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

Prolactin enhances T regulatory cell promotion of breast cancer through the long form prolactin receptor.

Previous work has shown systemic knockdown of the long form prolactin receptor (LFPRLR) in vivo markedly reduced metastasis in mouse models of breast cancer, but whether this translated to prolonged survival was unknown. Here we show that LFPRLR knockdown in the highly metastatic, immunocompetent 4T1 model prolonged survival and reduced recruitment of T regulatory cells (Tregs) to the tumor through effects on the production of CCL17. For the Tregs still recruited to the primary tumor, LFPRLR knockdown both directly and indirectly reduced their ability to promote tumor parenchymal epithelial to mesenchymal transition. Importantly, effects of prolactin on expression of mesenchymal genes by the tumor parenchyma were very different in the absence and presence of Tregs. While systemic knockdown of the LFPRLR downregulated transcripts important for immune synapse function in the remaining tumor Tregs, splenic Tregs seemed unaffected by LFPRLR knockdown, as demonstrated by their continued ability to suppress anti-CD3/CD28-stimulated effector cell proliferation at 1-5 months. These results demonstrate that knockdown of the LFPRLR achieves intra-tumor immunotherapeutic effects and suggest this occurs with reduced likelihood of peripheral inflammatory/autoimmune sequelae.

Sex Differences in the Immune System Become Evident in the Perinatal Period in the Four Core Genotypes Mouse.

We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

S179D Prolactin Sensitizes Human PC3 Prostate Cancer Xenografts to Anti-tumor Effects of Well-Tolerated Doses of Calcitriol.

Calcitriol has been shown to have multiple anti-prostate cancer effects both in vitro and in xenograft models, and associations between low levels of calcitriol and more aggressive forms of prostate cancer have been observed clinically. However, the concentrations of calcitriol required to have a substantive anti-cancer effect in vivo are toxic. In previous work, we had observed that the selective prolactin receptor modulator, S179D PRL, sensitized prostate cancer cells in vitro to physiological concentrations of calcitriol through an ability to increase expression of the vitamin D receptor. Here, we have investigated whether administration of S179D PRL would likewise sensitize androgen-insensitive human PC3 xenografts in vivo and do so without inducing tissue damage akin to hypervitaminosis D. Using low concentrations of both S179D PRL (250 ng/h) and calcitriol (up to 220 pg/h), we found no effect of each alone or in combination on the growth rate of tumors. However, there was increased central tumor death with their combination that was more than additive at 250 ng S179D PRL and 220 pg calcitriol per hour. Both S179D PRL and calcitriol alone were antiangiogenic, but their antiangiogenic effects were not additive. Also, both S179D PRL and calcitriol alone increased the number of apoptotic cells in tumor sections, but their combination reduced the number, suggesting more effective clearance of apoptotic cells. Histopathology of the livers and kidneys showed no changes consistent with hypervitaminosis D. We conclude that dual therapy holds promise as a means to harness the anti-tumor effects of well-tolerated doses of calcitriol.

Sex Differences in Mouse Popliteal Lymph Nodes.

Females have more robust immune responses than males, well-illustrated by the degree of inflammation elicited during delayed-type hypersensitivity (DTH) responses. Here, we have investigated underlying sex differences that may contribute to differential footpad DTH responses using wildtype and four core genotypes (FCG) mice and popliteal lymphnode cellularity and gene expression. DTH responses in XX and XY FCG females showed no role for almost all genes expressed on sex chromosomes. After then filtering-out genes differentially expressed between XX and XY females, only one gene was sexually differentially expressed in wildtype mice, glycosylation-dependent cell adhesion molecule 1 (Glycam1), expressed 7-fold higher in females. Glycam1 facilitates leukocyte entry through high endothelial venules. Consistent with greater Glycam1 expression, female nodes contained twice as many cells. While females had more memory T cells in their nodes, males had a higher percentage of T regulatory cells. This sexual dimorphism in wildtype animals manifested pre-pubertally, was enhanced post-pubertally, and was eliminated by castration. The formation of male gonads is determined by the expression of Sry. Sry overexpression, which does not affect testosterone levels, produced an exaggerated male phenotype. We conclude that Sry expression through formation of the male gonad indirectly negatively impacts the potential for local inflammation.

Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood.

Small oligonucleotides (oligos) are increasingly being utilized as diagnostics or treatments for disease. An impediment to broader use is the ability to readily measure oligos in biological fluids. Here, we describe a very straightforward assay with detection in the sub-picomole range that does not require extraction from serum/plasma or polymerization chain reaction amplification. As a result, there are no losses or errors due to sample handling, and the assay can be used to measure oligos modified in a variety of ways that increase therapeutic efficacy. The enzyme-linked oligonucleotide hybridization assay (ELOHA) is based on competition with a detection oligo for hybridization to a capture oligo covalently linked to a solid substrate. The versatility of ELOHAs is demonstrated by application to the measurement of three oligos, including two morpholino-oligos with 3'-octaguanidine derivatization for efficient cell uptake. The third oligo is unmodified and has a DNA sequence equivalent to miR93. The assays have sensitivity as low as 0.28 pmol/sample reaction at 50% hybridization. Adding to clinical utility is the need for only a simple 96-well absorbance plate reader and the finding that neither EDTA nor heparin interferes with detection.

Epitope-specific affinity maturation improved stability of potent protease inhibitory antibodies.

Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.

Use of a novel camelid-inspired human antibody demonstrates the importance of MMP-14 to cancer stem cell function in the metastatic process.

Matrix metalloproteinases (MMPs) are considered excellent targets for cancer therapy because of their important roles in multiple aspects of tumor growth and metastatic spread. However, not all MMPs, or even all activities of specific MMPs, promote cancer. Therefore, there is a need for highly specific inhibitors. Monoclonal antibodies provide the potential for the degree of specificity required, but the isolation of antibodies able to inhibit a specific protease with high selectivity is challenging. Proteolysis specificity lies in recognition of the substrate in or around the active site, which generally forms a concave cleft inaccessible by human IgGs. Inspired by camelid antibodies, which have convex paratopes, we have produced a recombinant human IgG, designated 3A2, which binds in the substrate cleft of MMP-14, inhibiting its activity, but not the activity of highly homologous MMPs. In the 4T1 highly metastatic, syngeneic, orthotopic model of breast cancer, IgG 3A2 markedly inhibited growth of the primary tumor, but more importantly reduced metastatic spread to the lungs and liver by 94%. Stem cells in the tumor population expressed twice as much MMP-14 mRNA as bulk tumor cells. In addition to reducing dissemination of tumor stem cells, as would be expected from inhibition of MMP-14's ability to degrade components of the extracellular matrix, IgG 3A2 also inhibited the ability of individual stem cells to proliferate and produce colonies. We conclude that it is possible to produce antibodies with sufficient specificity for development as therapeutics and that IgG 3A2 has therapeutic potential.

Lactation-Based Maternal Educational Immunity Crosses MHC Class I Barriers and Can Impart Th1 Immunity to Th2-Biased Recipients.

We have previously demonstrated lactational transfer of T cell-based immunity from dam to foster pup. In the short term, a significant part of transferred immunity is passive cellular immunity. However, as time progresses, this is replaced by what we have described as maternal educational immunity such that by young adulthood, all immune cells responding to a foster dam immunogen are the product of the foster pup's thymus. To reduce confounding factors, this original demonstration used congenic/syngeneic dam and foster pup pairs. In this study, we investigated lactational transfer of immunity to Mycobacterium tuberculosis in MHC class I-mismatched animals, as well as from Th1-biased dams to Th2-biased foster pups. Using immunized C57BL/6J dams, lactational transfer to nonimmunized BALB/cJ foster pups resulted in much greater immunity than direct immunization in 5-wk-old pups (ex vivo assay of pup splenocytes). At this age, 82% of immunogen-responding cells in the pup spleen were produced through maternal educational immunity. FVB/NJ nonimmunized foster recipients had a greater number of maternal cells in the spleen and thymus but a much larger percentage was Foxp3+, resulting in equivalent immunity to direct immunization. Depletion of maternal Foxp3+ cells from pup splenocytes illustrated a substantial role for lactationally transferred dam regulatory T cells in suppression of the ex vivo response in FVB/NJ, but not BALB/cJ, recipients. We conclude that lactational transfer of immunity can cross MHC class I barriers and that Th1 immunity can be imparted to Th2-biased offspring; in some instances, it can be greater than that achieved by direct immunization.

Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol.

Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3'UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3'UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3'UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3'UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17ß-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17ß-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17ß-estradiol miR-106b-elevating signaling pathways at ERα.

Role of Prolactin in Promotion of Immune Cell Migration into the Mammary Gland.

Immune cells in the mammary gland play a number of important roles, including protection against infection during lactation and, after passing into milk, modulation of offspring immunity. However, little is known about the mechanism of recruitment of immune cells to the lactating gland in the absence of infection. Given the importance of prolactin to other aspects of lactation, we hypothesized it would also play a role in immune cell recruitment. Prolactin treatment of adult female mice for a period equivalent to pregnancy and the first week of lactation increased immune cell flux through the mammary gland, as reflected in the number of immune cells in mammary gland-draining, but not other lymph nodes. Conditioned medium from luminal mammary epithelial HC11 cell cultures was chemo-attractive to CD4+ and CD8+ T cells, CD4+ and CD8+ memory T cells, B cells, macrophages, monocytes, eosinophils, and neutrophils. Prolactin did not act as a direct chemo-attractant, but through effects on luminal mammary epithelial cells, increased the chemo-attractant properties of conditioned medium. Macrophages and neutrophils constitute the largest proportion of cells in milk from healthy glands. Depletion of CCL2 and CXCL1 from conditioned medium reduced chemo-attraction of monocytes and neutrophils, and prolactin increased expression of these two chemokines in mammary epithelial cells. We conclude that prolactin is an important player in the recruitment of immune cells to the mammary gland both through its activities to increase epithelial cell number as well as production of chemo-attractants on a per cell basis.

Maternal Milk T Cells Drive Development of Transgenerational Th1 Immunity in Offspring Thymus.

Using multiple murine foster-nursing protocols, thereby eliminating placental transfer and allowing a distinction between dam- and pup-derived cells, we show that foster nursing by an immunized dam results in development of CD8(+) T cells in nonimmunized foster pups that are specific for Ags against which the foster dam was immunized (Mycobacterium tuberculosis or Candida albicans). We have dubbed this process "maternal educational immunity" to distinguish it from passive cellular immunity. Of the variety of maternal immune cells present in milk, only T cells were detected in pup tissues. Maternal T cells, a substantial percentage of which were CD4(+)MHC class II(+), accumulated in the pup thymus and spleen during the nursing period. Further analysis of maternal cells in the pup thymus showed that a proportion was positive for maternal immunogen-specific MHC class II tetramers. To determine the outcome of Ag presentation in the thymus, the maternal or foster pup origin of immunogen-responding CD8(+) cells in foster pup spleens was assessed. Whereas ∼10% were maternally derived in the first few weeks after weaning, all immunogen-responding CD8(+) T cells were pup derived by 12 wk of age. Pup-derived immunogen-responsive CD8(+) cells persisted until at least 1 y of age. Passive cellular immunity is well accepted and has been demonstrated in the human population. In this study, we show an arguably more important role for transferred immune cells: the direction of offspring T cell development. Harnessing maternal educational immunity through prepregnancy immunization programs has potential for improvement of infant immunity.

Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.