Plant Defensin γ-Thionin Induces MAPKs and Activates Histone Deacetylases in Bovine Mammary Epithelial Cells Infected With Staphylococcus aureus.

Defensins are an important group of host defense peptides. They have immunomodulatory properties, which have been mainly described for mammal defensins, but similar effects for plant defensins remain unknown. Previously, we showed that the defensin γ-thionin (Capsicum chinense) reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMECs) while inducing Toll-like receptor 2 (TLR2), modulating the inflammatory response. Here, we analyze the effect of γ-thionin on the TLR2 pathway in bMECs infected with S. aureus and determine if it modulates epigenetic marks. Pre-treated bMECs with γ-thionin (100 ng/ml) reduced the basal activation of p38 and ERK1/2 (~3-fold), but JNK was increased (~1.5-fold). Also, infected bMECs induced p38, but this effect was reversed by γ-thionin, whereas ERK1/2 was reduced by infection but stimulated by γ-thionin. Likewise, γ-thionin reduced the activation of Akt kinase ~50%. Furthermore, γ-thionin induced the activation of transcriptional factors of inflammatory response, highlighting EGR, E2F-1, AP-1, and MEF, which were turned off by bacteria. Also, γ-thionin induced the activation of histone deacetylases (HDACs, ~4-fold) at 24 h in infected bMECs and reduced LSD1 demethylase (HDMs, ~30%) activity. Altogether, these results demonstrated the first time that a plant defensin interferes with inflammatory signaling pathways in mammalian cells.

Prolactin and Estradiol are Epigenetic Modulators in Bovine Mammary Epithelial Cells during Staphylococcus aureus Infection.

Changes in the levels of reproductive hormones compromise the bovine innate immune response (IIR). Changes in 17ß-estradiol (E2) and prolactin (bPRL) levels affect the IIR of bovine mammary epithelial cells (bMECs), the target tissue of these hormones. In this work, we explored the effect of the combined hormones on bMEC IIR during Staphylococcus aureus infection, and if they can modulate epigenetic marks. By gentamicin protection assays, we determined that combined hormones (bPRL (5 ng/mL) and E2 (50 pg/mL)] decrease S. aureus internalization into bMECs (~50%), which was associated with a reduction in integrin α5ß1 membrane abundance (MA) (~80%) determined by flow cytometry. Additionally, combined hormones increased Toll-like receptor 2 (TLR2) MA (25%). By RT-qPCR, we showed that combined hormones induce the expression of pro- and anti-inflammatory cytokine genes, as well as up-regulate antimicrobial peptide gene expression. The combined hormones induced H3K9Ac at 12 h of treatment, which coincides with the reduction in histone deacetylase (HDAC, 15%) activity. In addition, hormones increased the H3K9me2 mark at 12 h, which correlates with a reduction in the expression of KDM4A. In conclusion, bPRL and E2 modulate the IIR of bMECs, an effect that can be related to the regulation of histone H3 modifications such as H3K9Ac and H3K9me2.

Immunomodulatory Effects of 17ß-Estradiol on Epithelial Cells during Bacterial Infections.

The innate immune system can function under hormonal control. 17ß-Estradiol (E2) is an important sexual hormone for the reproductive cycle of mammals, and it has immunomodulatory effects on epithelial cells, which are the first line of defense against incoming bacteria. E2 regulates various pathophysiological processes, including the response to infection in epithelial cells, and its effects involve the regulation of innate immune signaling pathways, which are mediated through estrogen receptors (ERs). E2 modulates the expression of inflammatory and antimicrobial elements such as cytokines and antimicrobial peptides. The E2 effects on epithelial cells during bacterial infections are characterized by an increase in the production of antimicrobial peptides and by the diminution of the inflammatory response to abrogate proinflammatory cytokine induction by bacteria. Here, we review several novel molecular mechanisms through which E2 regulates the innate immune response of epithelial cells against bacterial infections.

Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization.

17ß-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5ß1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1ß (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1ß. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization.

Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

ß-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal ß-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant ß-defensins remain unknown. In addition, the regulation of the immune system by mammalian ß-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized ß-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 µg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1ß (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of ß-defensins from plants as immunomodulators of the mammalian innate immune response.

The activation of the TLR2/p38 pathway by sodium butyrate in bovine mammary epithelial cells is involved in the reduction of Staphylococcus aureus internalization.

Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1ß and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen invasion, and NaB may exert anti-inflammatory effects during infection.

Non-classical effects of prolactin on the innate immune response of bovine mammary epithelial cells: Implications during Staphylococcus aureus internalization.

Staphylococcus aureus has the ability to invade mammary epithelial cells (bMECs) causing mastitis. This event depends primarily on the α5ß1 integrin in the host cell. In addition, bMECs are a target for the hormone prolactin (PRL), which can regulate ß1 integrin-dependent actions related to differentiation and lactation. Previously, we demonstrated that bovine PRL (bPRL, 5 ng/ml) stimulates S. aureus internalization into bMECs. TLR2 is important during S. aureus infections, but its activation by PRL has not yet been established. The objective of this study was to determine the role of α5ß1 integrin and TLR2 during S. aureus internalization into bMECs stimulated with bPRL. We demonstrated that the prolactin-stimulated internalization of S. aureus decreases in response to the blockage of α5ß1 integrin (∼ 80%) and TLR2 (∼ 80%). bPRL increases the membrane abundance (MA) of α5ß1 integrin (∼ 20%) and induces TLR2 MA (∼ 2-fold). S. aureus reduces the α5ß1 integrin MA in bMECs treated with bPRL (∼ 75%) but induces TLR2 MA in bMECs (∼ 3-fold). Bacteria and bPRL did not modify TLR2 MA compared with the hormone alone. S. aureus induces the activation of the transcription factor AP-1, which was inhibited in bMECs treated with bPRL and infected. In general, bPRL induces both pro- and anti-inflammatory responses in bMECs, which are abated in response to bacterial challenge. Interestingly, the canonical Stat-5 transcription factor was not activated in the challenged bMECs and/or treated with bPRL. Taken together, these results support novel functions of prolactin as a modulator of the innate immune response that do not involve the classical prolactin pathway.

Modulation of the inflammatory response of bovine mammary epithelial cells by cholecalciferol (vitamin D) during Staphylococcus aureus internalization.

Vitamin D is an immunomodulator that exerts anti-inflammatory effects. In this work, the effects of cholecalciferol, a vitamin D precursor, on the inflammatory response of bovine mammary epithelial cells (bMECs) during the internalization of Staphylococcus aureus were analyzed. Cholecalciferol and S. aureus inhibited TLR2 mRNA expression, but cholecalciferol differentially modulated the TLR2 membrane abundance. In fact, 50 nM cholecalciferol inhibited the TLR2 membrane abundance in bMECs infected with S. aureus, and this concentration also exerted the highest inhibitory effect on internalization. Cholecalciferol down-regulated the mRNA expression of TNF-α and IL-1ß and up-regulated that of RANTES and IL-10 but did not modify IL-6 and IL-8 expression. S. aureus strongly induced the mRNA expression of TNF-α, RANTES and IL-10 and inhibited IL-8 expression. Interestingly, cholecalciferol pre-treatments inhibited the bacterial-induced expression of TNF-α, IL-1ß, RANTES and IL-10. In conclusion, cholecalciferol differentially regulates the inflammatory response of bMECs during S. aureus internalization and may be an effective innate immunity modulator in mammary gland tissues.