Ameae M. Walker

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation in multiple organ systems. Although elevated circulating levels of the hormone/cytokine prolactin (PRL) have long been associated with increased SLE activity, the extent to which different isoforms of the prolactin receptor (PRLR) contribute to disease pathogenesis has not been explored. Here, we show that immune cells in SLE have aberrant splicing of the PRLR such that the ratio of the long (LF) to short (SF) splice isoforms is increased. To determine whether change in PRLR isoform expression was causal in this disease, we used a splice-modulating oligomer that knocks down expression of the LFPRLR while increasing expression of the SFPRLRs. Using patient samples ex vivo, SLE-prone mice in vivo, high-dimensional flow cytometry, and single-cell RNA sequencing, we demonstrated that the aberrant PRLR isoform expression in SLE both directly and indirectly contributes to the generation of autoreactive immune cell phenotypes. Accordingly, LFPRLR knockdown (KD) reduced STAT1 phosphorylation, decreased expression of type I interferon signaling/response genes known to be biomarkers and hub genes for SLE, reduced immunoglobulins with autoreactivity-associated sequence features, and averted glomerular pathology in SLE-prone mice. Importantly, LFPRLR KD reduced pathogenic B cell and other immune subsets that drive B cell activation, without negative impact on healthy donor counterparts. Current treatments for SLE adversely affect healthy cells and do not concurrently eradicate multiple pathogenic immune subsets. As LFPRLR splice-modulating oligomer does not share these disadvantages, KD of the LFPRLR represents a potential treatment strategy for SLE that merits further investigation.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation in multiple organ systems. While a clinical association between elevated levels of the hormone/cytokine, prolactin (PRL), and exacerbation of SLE has been recognized for some time, little is known about the mechanisms through which PRL affects the course of this disease. Here, we show that immune cells in SLE have aberrant splicing of the prolactin receptor (PRLR) such that the ratio of the long to short splice variants is increased. To determine whether the change in PRLR isoform expression was causal in this disease, we used a splice-modulating oligomer (SMO) that knocked down expression of the long splice variant (LFPRLR). Using patient samples ex vivo , SLE-prone mice in vivo , high-dimensional flow cytometry and single-cell RNA-sequencing, we demonstrate that the aberrant PRLR isoform expression in SLE both directly and indirectly drives production of autoreactive immune cell phenotypes. Thus, LFPRLR knockdown decreased the expression of type I interferon signaling/response genes known to be biomarkers and hub genes for SLE, reduced immunoglobulins with signatures considered autoreactive, and averted glomerular kidney damage in SLE-prone mice. Importantly, LFPRLR knockdown reduced pathogenic B cells and other immune subsets that drive B-cell activation, without negative impact on healthy donor counterparts. Current treatments for SLE adversely affect healthy cells and do not concurrently eradicate multiple pathogenic immune subsets. Since LFPRLR SMO does not share these disadvantages, knockdown of the LFPRLR represents a potential treatment strategy for SLE that merits further investigation. Graphical The LFPRLR represents an attractive therapeutic target in SLE. ( Left ) In addition to increased pituitary/circulating PRL, individuals with SLE exhibit aberrant increases in the production of autocrine/paracrine PRL by immune cells, and in their expression of specifically the long isoform (LF) of the PRLR. ( Middle ) Expression of the LFPRLR specifically enhances autoreactive immunophenotypes and promotes lupus nephritis. ( Right ) A splice modulating oligomer (SMO), that prevents synthesis of only the LFPRLR but not the short PRLR isoforms, reduces pathogenic immunophenotypes without affecting the normal counterparts of immune cells.

Using four core genotypes (FCG) mice, we have previously shown a larger number of CD4+ and CD8+ T cells in the spleens of female mice, a sex difference that develops by postnatal day 7 and is retained through adulthood. This difference in splenic T cell number is a consequence of reduced thymic egress and reduced splenic seeding in male mice, caused in part by the male-specific perinatal surge of testosterone, and in part by Sry, which is overexpressed in this model. Here, we used the background strain for FCG mice (C57BL/6J) to ask whether sex influenced actual immunity in the postnatal period. Pups were immunized on postpartum days 1 or 3 with Mycobacterium tuberculosis (Mtb), challenged on day 7 with Mtb purified protein derivative (PPD), and sacrificed on day 8. Subsequent ex vivo challenges of splenocytes showed PPD-stimulated CD8+ responses (increased CD8+, increased CD8+CD44hi, decreased CD8+CD44hiCD127-/lo) but no differences between males and females. However, when CD8+ T cells were analyzed for IFN-γ and IL-2 production, although there was no sex difference in mono-functional IFN-γ+ (100%) or IL-2+ (67%), only females (0% of males and 42% of females) produced bi-functional (IFN-γ+IL-2+) cells. Ex vivo PPD-stimulated responses of other relevant cells from the spleen showed no sex differences in dendritic cells (CD11c+CD86+IL-6+) but females had more (3-fold) IL-6-producing macrophages (F4/80+CD86+IL-6+) and reduced T regulatory cells (CD4+CD25+Foxp3+). We conclude that some sex differences in immunity are evident at one week of age in Mtb immunized mouse pups, with females exhibiting qualitatively superior Mtb-specific immune responses.

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.

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.

A proportion of secreted pituitary prolactin (PRL) is phosphorylated. However, because most commercial sources of PRL are recombinant proteins without posttranslational modification, the importance of PRL phosphorylation to the production of milk proteins is an understudied area. Here, we have examined the effect of PRL phosphorylation on expression of the milk protein, β-casein, using a phospho-stable mimic of the phosphorylated form (S179D-PRL) and analyzing promoter activation and mRNA stability over a 7-day treatment period in response to this and unmodified PRL. At equivalent concentrations, the phospho-mimic showed a superior ability to activate a −2300 → +490 region of the promoter, but not an artificial promoter consisting of three Stat5 consensus sites upstream of a minimal promoter. Unlike unmodified PRL, S179D-PRL was also able to stabilize β-casein mRNA. These effects of S179D-PRL were eliminated by incubation in the MAP kinase pathway inhibitor, U0126, bringing promoter activation down to the level seen with unmodified PRL and essentially eliminating the effect on mRNA stability. These results support an important role for the posttranslational phosphorylation of PRL and signaling through the MAP kinase pathway in the production of this milk protein.

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.

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.