Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

TRPV4 increases cardiomyocyte calcium cycling and contractility yet contributes to damage in the aged heart following hypoosmotic stress.

Aims: Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress. Methods and results: TRPV4 protein expression increased in cardiomyocytes of Aged (24-27 months) mice compared with Young (3-6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 µmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1 µmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage. Conclusions: Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

Increased susceptibility to OVX-associated metabolic dysfunction in UCP1-null mice.

Premenopausal females are protected against adipose tissue inflammation and insulin resistance, until loss of ovarian hormone production (e.g., menopause). There is some evidence that females have greater brown adipose tissue (BAT) thermogenic capacity. Because BAT mass correlates inversely with insulin resistance, we hypothesized that increased uncoupling protein 1 (UCP1) expression contributes to the superior metabolic health of females. Given that UCP1 transiently increases in BAT following ovariectomy (OVX), we hypothesized that UCP1 may 'buffer' OVX-mediated metabolic dysfunction. Accordingly, female UCP1 knock-out (KO) and wild-type (Digby, et al.) mice received OVX or sham (SHM) surgeries at 12 weeks of age creating four groups (n=10/group), which were followed for 14 weeks and compared for: body weight and adiposity, food intake, energy expenditure and spontaneous physical activity (metabolic chambers), insulin resistance (HOMA-IR, ADIPO-IR, and glucose tolerance testing), and adipose tissue phenotype (histology, gene, and protein expression). Two-way ANOVA was used to assess main effects of genotype (G), OVX treatment (O), and genotype by treatment (GxO) interactions, which were considered significant when P<0.05. UCP1KO mice experienced a more adverse metabolic response to OVX than WT. Whereas OVX-induced weight gain was not synergistically greater for KO compared to WT (GxO, NS), OVX-induced insulin resistance was significantly exacerbated in KO compared to WT (GxO for HOMA-IR, P<0.05). These results suggest UCP1 is protective against metabolic dysfunction associated with loss of ovarian hormones and support the need for more research into therapeutics to selectively target UCP1 for prevention and treatment of metabolic dysfunction following ovarian hormone loss.

Deletion of UCP1 enhances ex vivo aortic vasomotor function in female but not male mice despite similar susceptibility to metabolic dysfunction.

Females are typically more insulin sensitive than males, which may be partly attributed to greater brown adipose tissue (BAT) activity and uncoupling protein 1 (UCP1) content. Accordingly, we tested the hypothesis that UCP1 deletion would abolish sex differences in insulin sensitivity and that whitening of thoracic periaortic BAT caused by UCP1 loss would be accompanied with impaired thoracic aortic function. Furthermore, because UCP1 exerts antioxidant effects, we examined whether UCP1 deficiency-induced metabolic dysfunction was mediated by oxidative stress. Compared with males, female mice had lower HOMA- and AT-insulin resistance (IR) despite no significant differences in BAT UCP1 content. UCP1 ablation increased HOMA-IR, AT-IR, and whitening of BAT in both sexes. Expression of UCP1 in thoracic aorta was greater in wild-type females compared with males. Importantly, deletion of UCP1 enhanced aortic vasomotor function in females only. UCP1 ablation did not promote oxidative stress in interscapular BAT. Furthermore, daily administration of the free radical scavenger tempol for 8 wk did not abrogate UCP1 deficiency-induced increases in adiposity, hyperinsulinemia, or liver steatosis. Collectively, we report that 1) in normal chow-fed mice housed at 25°C, aortic UCP1 content was greater in females than males and its deletion improved ex vivo aortic vasomotor function in females only; 2) constitutive UCP1 content in BAT was similar between females and males and loss of UCP1 did not abolish sex differences in insulin sensitivity; and 3) the metabolic disruptions caused by UCP1 ablation did not appear to be contingent upon increased oxidative stress in mice under normal dietary conditions.

Loss of UCP1 exacerbates Western diet-induced glycemic dysregulation independent of changes in body weight in female mice.

We tested the hypothesis that female mice null for uncoupling protein 1 (UCP1) would have increased susceptibility to Western diet-induced "whitening" of brown adipose tissue (AT) and glucose intolerance. Six-week-old C57BL/6J wild-type (WT) and UCP1 knockout (UCP1-/-) mice, housed at 25°C, were randomized to either a control diet (10% kcal from fat) or Western diet (45% kcal from fat and 1% cholesterol) for 28 wk. Loss of UCP1 had no effect on energy intake, energy expenditure, spontaneous physical activity, weight gain, or visceral white AT mass. Despite similar susceptibility to weight gain compared with WT, UCP1-/- exhibited whitening of brown AT evidenced by a striking ~500% increase in mass and appearance of large unilocular adipocytes, increased expression of genes related to inflammation, immune cell infiltration, and endoplasmic reticulum/oxidative stress (P < 0.05), and decreased mitochondrial subunit protein (COX I, II, III, and IV, P < 0.05), all of which were exacerbated by Western diet (P < 0.05). UCP1-/- mice also developed liver steatosis and glucose intolerance, which was worsened by Western diet. Collectively, these findings demonstrate that loss of UCP1 exacerbates Western diet-induced whitening of brown AT, glucose intolerance, and induces liver steatosis. Notably, the adverse metabolic manifestations of UCP1-/- were independent of changes in body weight, visceral adiposity, and energy expenditure. These novel findings uncover a previously unrecognized metabolic protective role of UCP1 that is independent of its already established role in energy homeostasis.

Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library.

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 microM. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.