Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity

<div><p>The traditional Chinese medicine Chan-Su is widely used for treatment of cancer and cardiovascular diseases, but also as a remedy for infections such as furunculosis, tonsillitis and acute pharyngitis. The clinical use of Chan-Su suggests that it has anti-infective effects, however, the mechanism of action is incompletely understood. In particular, the effect on the human immune system is poorly defined. Here, we describe previously unrecognized immunomodulatory activities of cinobufagin (CBG), a major bioactive component of Chan-Su. Using human monocyte-derived dendritic cells (DCs), we show that LPS-induced maturation and production of a number of cytokines was potently inhibited by CBG, which also had a pro-apoptotic effect, associated with activation of caspase-3. Interestingly, CBG triggered caspase-1 activation and significantly enhanced IL-1β production in LPS-stimulated cells. Finally, we demonstrate that CBG upregulates gene expression of the antimicrobial peptides (AMPs) hBD-2 and hBD-3 in DCs, and induces secretion of HNP1-3 and hCAP-18/LL-37 from neutrophils, potentiating neutrophil antibacterial activity. Taken together, our data indicate that CBG modulates the inflammatory phenotype of DCs in response to LPS, and triggers an antibacterial innate immune response, thus proposing possible mechanisms for the clinical effects of Chan-Su in anti-infective therapy.</p></div

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract-4

points. B) CRS peptides and cryptdins measured by Western blot from four individual BALB/c after three days of Listeria infection (denoted by triangles). Four mice receiving PBS were used for comparison (squares).<p><b>Copyright information:</b></p><p>Taken from "Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract"</p><p>http://www.biomedcentral.com/1471-2172/9/37</p><p>BMC Immunology 2008;9():37-37.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2488317.</p><p></p

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract-1

RNA copy numbers were determined in the duodenum, jejunum, and ileum. The "x" numbers denote relative copy numbers between ileum and duodenum. The results are based on four FVB mice, and are given as mean and range. B) Comparative distributions in duodenum and ileum. Of note is that duodenum has a total of 2 × 10copies of Paneth cell products, while ileum has 18 × 10copies (area of the smaller duodenal and the ileal pies are proportional to total mRNA copy counts of analyzed antimicrobials). Colours denote individual Paneth cell products. C) Western blot of extracts from two individual BALB/c mice was analyzed using antibodies to CRS4C-3 and cryptdin 2. Duod = duodenum, Jej = jenunum and Ile = ileum.<p><b>Copyright information:</b></p><p>Taken from "Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract"</p><p>http://www.biomedcentral.com/1471-2172/9/37</p><p>BMC Immunology 2008;9():37-37.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2488317.</p><p></p

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract-0

Nification of the base of the crypt is shown in (B). Hoechst staining (blue) was used to visualize the nucleus.<p><b>Copyright information:</b></p><p>Taken from "Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract"</p><p>http://www.biomedcentral.com/1471-2172/9/37</p><p>BMC Immunology 2008;9():37-37.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2488317.</p><p></p

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract-2

S were determined in the duodenum and ileum at post-natal days 14 and 18 (preweaning) and adult mice. Means and range are depicted. The "x" numbers denote relative copy numbers measured by qRT-PCR between adult and 14 day old mice.<p><b>Copyright information:</b></p><p>Taken from "Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract"</p><p>http://www.biomedcentral.com/1471-2172/9/37</p><p>BMC Immunology 2008;9():37-37.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2488317.</p><p></p

Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract-3

E depicted. Numbers denotes relative copy numbers between small intestine of germ-free and conventional NMRI/KI mice. Four mice in each group. * denotes P < 0,03.<p><b>Copyright information:</b></p><p>Taken from "Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract"</p><p>http://www.biomedcentral.com/1471-2172/9/37</p><p>BMC Immunology 2008;9():37-37.</p><p>Published online 17 Jul 2008</p><p>PMCID:PMC2488317.</p><p></p

Inhibited cytokine release of LPS-stimulated DCs.

<p>DCs were stimulated with LPS (100 ng/ml) in the presence of vehicle or CBG for 24 hours. Supernatants were collected and analyzed for IL-6, IL-8, IL-12p40, TNF-α and IL-10. Data shown represent cytokine production for each donor and mean values for 6–8 donors. *P<0.05. **P<0.01.</p

Univariable logistic regression with treatment-related factors during chemotherapy as independent variables and oral mucositis as the outcome in all patients (n = 104).

<p>OR, odds ratio; CI, confidence interval.</p>a<p>Variables with OR <1 were not included in multivariable analysis since cytostatic regimens can only be considered as risk indicators for oral mucositis.</p>*<p><i>P</i><0.05,</p>**<p><i>P</i><0.01.</p

Induction of AMPs in DCs and neutrophils.

<p>(A) DCs were stimulated with vehicle or CBG in the absence or presence of LPS for 24 hours. Quantitative polymerase chain reaction (qPCR) for hBD-2 and hBD-3, normalized to GAPDH, was performed, and data shown represent mean gene expression (fold change compared to vehicle) + SEM for 3 donors. (B-D) Neutrophils were stimulated with vehicle or CBG in the absence or presence of LPS for 6 hours. (B) Neutrophil supernatants analyzed for release of HNP1-3 by ELISA. Cytochalasin B and fMLP (c/fMLP) treated cells served as positive control. Data shown represent means + SEM for 3 donors. *P<0.05. **P<0.01. (C) Neutrophil supernatants analyzed for release of proform hCAP-18 by semi-quantitative Western blot. Cytochalasin B/ fMLP (c/fMLP) served as positive control. Human serum containing 1 μg/ml hCAP-18 was used as a reference for relative determination of supernatant hCAP-18 levels. One representative donor out of two is shown (left) and data represent means + SD for 2 donors (right). (D) Bacterial killing was measured following incubation of neutrophils with live <i>S</i>. <i>pneumoniae</i> (strain T4R) at an MOI of 0.1 for 1 hour. Percentage live bacteria was calculated based on colony forming units (CFU) per ml obtained relative to vehicle-treated neutrophils. Data shown represent mean + SEM for 3 donors. *P<0.05.</p

CBG triggers IL-1β production and caspase-1 activation.

<p>DCs were stimulated with vehicle or CBG in the absence or presence of LPS (100 ng/ml) for 24 hours. (A) Supernatants were collected and analyzed for IL-1β production. Data shown represent means + SEM of IL-1β production for 17 donors. (B) Inflammasome activation was examined by analyzing the percentage of caspase-1 positive cells. Data shown represent means + SEM for 3 donors. (C) Whole-cell extracts were prepared, and equal amounts of lysates were analyzed by Western blot using an antibody against caspase-1. β-actin was used as a loading control. The results shown are representative of three independent experiments. (D) Cells were pretreated with the caspase-1 inhibitor Z-YVAD-FMK for 1 hour. Supernatants were collected at 24 hours and analyzed for IL-1β production. Data shown represent means + SEM of IL-1β production for 3 donors. **P<0.01. *P<0.05.</p