Placentas associated with female neonates from pregnancies complicated by urinary tract infections have higher cAMP content and cytokines expression than males.

PROBLEM: The cAMP pathway is involved in important biological processes including immune regulation and hormone signaling. At the feto-maternal unit, cAMP participates in placental function/physiology and the establishment of immunoendocrine networks. Low cAMP in male fetuses cord blood has been linked to poorer perinatal outcomes; however, cAMP placental content and its relationship with immune factors and fetal sex in an infectious condition have not been investigated. METHOD OF STUDY: Sex-dependent changes in cAMP content and its association with cytokines and antimicrobial peptides expression were studied in human placentas collected from normal pregnancies and with urinary tract infections (UTI). Radioimmunoassay was used to quantify cAMP in placental tissue, while immune markers expression was studied by qPCR. Additionally, cAMP effect on antimicrobial peptides expression was studied in cultured trophoblasts challenged with lipopolysaccharide, to mimic an infection. RESULTS: In UTI, placentas from female neonates had higher cAMP tissue content and increased expression of TNFA, IL1B, and IL10 than those from males, where IFNG was more elevated. While cAMP negatively correlated with maternal bacteriuria and IFNG, it positively correlated with the antimicrobial peptide S100A9 expression in a sex-specific fashion. In cultured trophoblasts, cAMP significantly stimulated ß-defensin-1 while reduced the lipopolysaccharide-dependent stimulatory effect on ß-defensin-2, ß-defensins-3, and S100A9. CONCLUSION: Our results showed higher cAMP content and defense cytokines expression in placentas associated with female neonates from pregnancies complicated by UTI. The associations between cAMP and bacteriuria/immune markers, together with cAMP's ability to differentially regulate placental antimicrobial peptides expression, suggest a dual modulatory role for cAMP in placental immunity.

Synergistic Antitumorigenic Activity of Calcitriol with Curcumin or Resveratrol is Mediated by Angiogenesis Inhibition in Triple Negative Breast Cancer Xenografts.

Calcitriol is a multitarget anticancer hormone; however, its effects on angiogenesis remain contradictory. Herein, we tested whether the antiangiogenic phytochemicals curcumin or resveratrol improved calcitriol antitumorigenic effects in vivo. Triple-negative breast cancer tumoral cells (MBCDF-T) were xenografted in nude mice, maintaining treatments for 3 weeks. Tumor onset, volume and microvessel density were significantly reduced in mice coadministered with calcitriol and curcumin (Cal+Cur). Vessel count was also reduced in mice simultaneously treated with calcitriol and resveratrol (Cal+Rsv). Cal+Cur and Cal+Rsv treatments resulted in less tumor activated endothelium, as demonstrated by decreased tumor uptake of integrin-targeted biosensors in vivo. The renal gene expression of Cyp24a1 and Cyp27b1 suggested increased calcitriol bioactivity in the combined regimens. In vitro, the phytochemicals inhibited both MBCDF-T and endothelial cells proliferation, while potentiated calcitriol's ability to reduce MBCDF-T cell-growth and endothelial cells migration. Resveratrol induced endothelial cell death, as deduced by increased sub-G1 cells accumulation, explaining the reduced tumor vessel number in resveratrol-treated mice, which further diminished when combined with calcitriol. In conclusion, the concomitant administration of calcitriol with curcumin or resveratrol synergistically promoted anticancer effects in vitro and in vivo in human mammary tumor cells. Whereas the results suggest different mechanisms of action of the phytochemicals when coadministered with calcitriol, the converging biological effect was inhibition of tumor neoangiogenesis.

Negative correlation between testosterone and TNF-α in umbilical cord serum favors a weakened immune milieu in the human male fetoplacental unit.

Clinical and epidemiological evidence supports that pregnancies carrying a male fetus are more vulnerable to infections and preterm birth, probably due to testosterone immunosuppressive properties. In human placentas, testosterone lowers the expression of CYP27B1, the vitamin D (VD)-activating enzyme, diminishing cathelicidin synthesis, a potent VD-dependent antimicrobial peptide (AMP). VD also stimulates other AMPs, including defensins. To get insights into the increased male vulnerability mechanisms, we investigated the relationship between fetal sex and the immunoendocrine milieu at the fetoplacental unit. For this, umbilical vein serum and placental samples were collected from healthy newborns. In males' serum, testosterone levels were significantly higher and negatively associated with TNF-α, a cytokine that strengthens the immune response. Males showed lower serum TNF-α and increased levels and gene expression of the immunosuppressive cytokine IL-10. Only in female samples there was a positive association (P < 0.05) between AMPs and both TNF-α and CYP27B1 and between 25-hydroxyvitamin D3 and IL-1ß serum levels. Accordingly, VD-metabolites (25-hydroxyvitamin D3, calcitriol) significantly stimulated IL-1ß gene expression in cultured trophoblasts. Interestingly, IL-1ß mRNA correlated positively with defensins (P < 0.05) in males, but not with cathelicidin expression, which was significantly diminished in comparison to females. Our data suggest that high umbilical serum testosterone and IL-10 in males could explain reduced TNF-α levels and lack of association between VD-dependent innate immunity markers and proinflammatory cytokines expression in the fetoplacental unit. Altogether, our observations imply a restricted basal immune milieu in males compared to females, which may help understand the higher male susceptibility to adverse perinatal outcomes.

Lipopolysaccharide and cAMP modify placental calcitriol biosynthesis reducing antimicrobial peptides gene expression.

PROBLEM: Calcitriol, the hormonal form of vitamin D3 (VD), stimulates placental antimicrobial peptides expression; nonetheless, the regulation of calcitriol biosynthesis in the presence of bacterial products and its consequence on placental innate immunity have scarcely been addressed. METHOD OF STUDY: We investigated how some bacterial products modify placental VD metabolism and its ability to induce antimicrobial peptides gene expression. RESULTS: Cultured human trophoblasts biosynthesized calcitriol only in the presence of its precursor calcidiol, a process that was inhibited by cyclic-AMP but stimulated by lipopolysaccharide (LPS). Intracrine calcitriol upregulated cathelicidin, S100A9, and ß-defensins (HBDs) gene expression, while LPS further stimulated HBD2 and S100A9. Unexpectedly, LPS significantly repressed cathelicidin basal mRNA levels and drastically diminished calcidiol ability to induce it. Meanwhile, cyclic-AMP, which is used by many microbes to avoid host defenses, suppressed calcitriol biosynthesis, resulting in significant inhibition of most VD-dependent microbicidal peptides gene expression. CONCLUSION: While LPS stimulated calcitriol biosynthesis, cyclic-AMP inhibited it. LPS downregulated cathelicidin mRNA expression, whereas cyclic-AMP antagonized VD-dependent-upregulation of most antimicrobial peptides. These findings reveal LPS and cyclic-AMP involvement in dampening placental innate immunity, highlighting the importance of cyclic-AMP in the context of placental infection and suggesting its participation to facilitate bacterial survival.

Selective immuno-modulatory effect of prolactin upon pro-inflammatory response in human fetal membranes.

During pregnancy, prolactin (PRL) is a neuro-immuno-cytokine that contributes actively to the crosstalk between the immune and endocrine systems and, thus, to the creation of an immune-privileged milieu. This work aims to analyze the capacity of PRL to modulate the synthesis and secretion of pro-inflammatory markers associated with labor. Studies were conducted using human fetal membranes at term mounted in a model of two independent chambers. The choriodecidual region was stimulated with 500-ng/mL lipopolysaccharide (LPS), and the amnion and choriodecidual region were co-simulated with different concentrations of PRL that can arise during pregnancy: 250, 500, 1000, and 4000ng/mL. Following these co-treatments, the tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-10 levels were measured in both compartments. As expected, treatment with LPS induced all cytokines to increase. Co-stimulation with the highest tested concentration of PRL induced significant decreases in TNF-α in the choriodecidual region and IL-1ß in both regions of the fetal membranes. PRL did not modified the IL-6 and IL-10 secretion profile. These findings, coupled with clinical evidence, suggest that the high level of PRL in the amniotic cavity is involved the mechanism by which the fetal-placental unit regulates the equilibrium between pro- and anti-inflammatory modulators.

Chronic moderate ethanol intake differentially regulates vitamin D hydroxylases gene expression in kidneys and xenografted breast cancer cells in female mice.

Factors affecting vitamin D metabolism may preclude anti-carcinogenic effects of its active metabolite calcitriol. Chronic ethanol consumption is an etiological factor for breast cancer that affects vitamin D metabolism; however, the mechanisms underlying this causal association have not been fully clarified. Using a murine model, we examined the effects of chronic moderate ethanol intake on tumoral and renal CYP27B1 and CYP24A1 gene expression, the enzymes involved in calcitriol synthesis and inactivation, respectively. Ethanol (5% w/v) was administered to 25-hydroxyvitamin D3-treated or control mice during one month. Afterwards, human breast cancer cells were xenografted and treatments continued another month. Ethanol intake decreased renal Cyp27b1 while increased tumoral CYP24A1 gene expression.Treatment with 25-hydroxyvitamin D3 significantly stimulated CYP27B1 in tumors of non-alcohol-drinking mice, while increased both renal and tumoral CYP24A1. Coadministration of ethanol and 25-hydroxyvitamin D3 reduced in 60% renal 25-hydroxyvitamin D3-dependent Cyp24a1 upregulation (P<0.05). We found 5 folds higher basal Cyp27b1 than Cyp24a1 gene expression in kidneys, whereas this relation was inverted in tumors, showing 5 folds more CYP24A1 than CYP27B1. Tumor expression of the calcitriol target cathelicidin increased only in 25-hydroxyvitamin D3-treated non-ethanol drinking animals (P<0.05). Mean final body weight was higher in 25-hydroxyvitamin D3 treated groups (P<0.001). Overall, these results suggest that moderate ethanol intake decreases renal and tumoral 25-hydroxyvitamin D3 bioconversion into calcitriol, while favors degradation of both vitamin D metabolites in breast cancer cells. The latter may partially explain why alcohol consumption is associated with vitamin D deficiency and increased breast cancer risk and progression.

IL-10 inhibits while calcitriol reestablishes placental antimicrobial peptides gene expression.

IL-10 and calcitriol help to achieve a successful pregnancy by suppressing active maternal immunity; however, these factors exert opposite effects upon microbial infections. In the skin and immune cells, IL-10 downregulates ß-defensins while calcitriol induces cathelicidin gene expression in various tissues including placenta. Though, the regulation of human placental ß-defensins by IL-10 and calcitriol has not been studied. Therefore, we explored the regulation of these antimicrobial peptides expression in cultured placental cells by calcitriol and IL-10 alone and combined. Real time PCR showed that calcitriol stimulated, while IL-10 inhibited, ß-defensins and cathelicidin gene expression (P<0.05). In coincubations studies, calcitriol was able to maintain antimicrobial peptides gene expression above control values, overriding IL-10 inhibitory effects. Calcitriol downregulated endogenous IL-10 secretion. Interestingly, calcitriol and TNF-α cooperatively enhanced ß-defensins, while TNF-α reduced basal and calcitriol-stimulated cathelicidin gene expression. In summary, calcitriol and IL-10 exerted opposite effects on antimicrobial peptides expression in the human placenta, suggesting that unbalanced production of IL-10 and calcitriol could be deleterious to innate immune responses during gestation. Our results suggest that calcitriol enhancement of placental defenses involves two mechanisms: (1) downregulation of IL-10 secretion and (2) direct upregulation of ß-defensins and cathelicidin gene expression. Considering that IL-10 and calcitriol differentially regulate the innate immune response in the placenta, in the case of an infection, calcitriol might restrict IL-10 permissive actions towards microbial invasion while restrains inflammation, allowing for pregnancy to continue in quiescence. These results strongly advice maternal vitamin D sufficiency during pregnancy.