The authors have declared that no competing interests exist.
Conceived and designed the experiments: SY MK FS. Performed the experiments: SY JOL KN. Analyzed the data: SY SS FS. Contributed reagents/materials/analysis tools: JOL DNH. Wrote the paper: SY MK FS.
In our previous studies, peripheral blood lineage−CD34+CD31+ cells (CD31+ IMC) appearing in severely burned patients have been characterized as inhibitor cells for the production of β-defensins (HBDs) by human epidermal keratinocytes (NHEK). In this study, the effect of glycyrrhizin on pseudomonal skin infections was studied in a chimera model of thermal injury. Two different chimera models were utilized. Patient chimeras were created in murine antimicrobial peptide-depleted NOD-SCID IL-2rγnull mice that were grafted with unburned skin tissues of severely burned patients and inoculated with the same patient peripheral blood CD31+ IMC. Patient chimera substitutes were created in the same mice that were grafted with NHEK and inoculated with experimentally induced CD31+ IMC. In the results, both groups of chimeras treated with glycyrrhizin resisted a 20 LD50 dose of
Previously, we have reported that glycyrrhizin protects severely burned mice from lethal doses of
Glycyrrhizin, a component of licorice root, has been clinically utilized in patients with peptic ulcers, chronic hepatitis, and cirrhosis. Glycyrrhizin has been reported to stimulate various host defense immunities, including inhibition of inflammatory responses
In the results presented herein, antimicrobial peptides were not produced significantly at grafted site skin tissues in patient chimeras, while the peptides were produced in the same chimeras treated with glycyrrhizin. The compound did not directly stimulate the production and mRNA expression of HBD-1 by epidermal keratinocytes. Sepsis stemming from
All animal experiments were performed in accordance with protocols approved by The Institutional Animal Care and Use Committee of The University of Texas Medical Branch at Galveston, TX (IACUC approval number: 0404019A). The study was approved by the Institutional Review Board of the University of Texas Medical Branch (IRB approved number: 02-018). Written informed consent for blood sampling was obtained from all adult subjects. For blood sampling from children, written parental consent was obtained. Ethical approval was obtained from the Ethical and Scientific Committee of the University of Texas Medical Branch.
Nine- to 12-week-old male NOD-SCID IL-2rγ−/− mice (NOD.Cg-
Eighteen burn patients (11 male, 7 female) admitted to the Shriners Hospitals for Children at Galveston and the University of Texas Medical Branch (UTMB) were enrolled in this study. All patients had more than a 30% total body surface area (TBSA) burn (average 56.3±18.2%). The youngest and oldest ages were 2 and 41 years old (average 11.2±9.3), respectively. All patients were subjected to a standard treatment regimen
HBD-1 kits were purchased from PeproTech (Rocky Hill, NJ, USA). HBD-1, MBD-1, anti-HBD-1, and anti-MBD-1 antibodies were purchased from Alpha Diagnostic International (San Antonio, TX, USA). Lineage cell depletion kits were purchased from Miltenyi Biotec (Auburn, CA, USA). FITC-conjugated anti-CD34 mAb and PE-conjugated anti-CD31 mAb were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and BioLegend (San Diego, CA, USA), respectively. Recombinant (r) CCL2 and rIL-10 were obtained from PeproTech, and mAbs directed against CCL2 and IL-10 were purchased from BioLegend. Adult normal human epidermal keratinocytes (NHEK) were obtained from Lonza (Walkersville, MD, USA) and propagated in a serum-free keratinocyte growth medium (KMG-2, Lonza) at 37°C. NHEK that underwent a second passage with KMG-2 were stored in liquid nitrogen. NHEK grown from the stored cells (the third passage) were used in this study. RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) was used as culture media for lineage−CD34+ cells. A Protease Inhibitor Cocktail was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Glycyrrhizin (20β-carboxy-11-oxo-30-norolean-12-en-3β-yl-2-
Lineage−CD34+CD31+ cells (patient CD31+ IMC) were prepared from peripheral mononuclear cells of severely burned patients, as previously described
γ-Irradiated NOD-SCID IL-2rγ−/− mice were grafted with unburned skin tissue removed from burn patients (discarded skin from the auto-graft surgery). The function of these discarded skin tissues to produce HBD-1 has been demonstrated
(1)
(2)
To determine the protective effect of glycyrrhizin against
Data shown in each figure are derived from three independent experiments using different donors. Data are presented as mean ± SEM. Survival curves were analyzed using the Kaplan-Meier method. Chimera groups were statistically compared using a Student’s
The importance of β-defensins on host resistance against
Patient chimeras created with unburned skin tissues from patients #1∼#3 and their respective CD31+ IMC were treated twice (6 and 24 hours after the IMC inoculation, i.p.) with glycyrrhizin. One day after the final treatment, 5 skin biopsies obtained from the chimera’s skin grafted sites were homogenized together in 1 ml PBS and assayed for HBD-1 by ELISA. As a result, HBD-1 was not detected in skin samples of any patient chimeras. However, peptide was detected in the chimeras treated with 10 mg/kg of glycyrrhizin at levels produced in γ-irradiated NOD-SCID IL-2rγ−/− mice grafted with patient unburned tissues (
After treatment with or without glycyrrhizin (10 mg/kg, i.p., twice), survival of patient chimera substitutes i.d. infected with 20 LD50 of
The effect of glycyrrhizin on the suppressor cell activity of patient CD31+ IMC on HBD-1 production by patient unburned skin tissues was examined
In addition, amounts of HBD-1 in the culture fluids obtained from the above transwell-cultures were measured by ELISA. HBD-1 production by unburned skin tissues from burn patients #11∼#14 was inhibited when it was transwell-cultured with patient CD31+ IMC. However, HBD-1 was produced when the same transwell-cultures were performed with 100 µg/ml of glycyrrhizin (
In our accompanying paper
Patient #16∼#18 CD31+ IMC (1×106 cells/ml) were individually treated with or without glycyrrhizin (100 µg/ml) for 12 hours. After washing with media, these cells were cultured for an additional 36 hours. Culture fluids harvested from the cultures supplemented with or without glycyrrhizin were added (5∼20%, v/v) to cultures of patient unburned skin tissues (0.2×0.2 cm), and cultured for 48 hours. Culture fluids harvested were assayed for HBD-1 by ELISA. *
In our accompanying paper
CCL2 (
Severely burned patients with decreased host antibacterial defenses are highly susceptible to various infections
Based on our previous studies
Currently, intravenous doses of glycyrrhizin (80–120 mg/day) is clinically utilized in patients with chronic hepatitis
Glycyrrhizin did not have any direct action on the growth of
How glycyrrhizin suppresses cytokine production by CD31+ IMC is not known. Recent studies have indicated that glycyrrhizin binds to the glucocorticoid-like receptor
CD31+ IMC may function to suppress the burn-associated inflammation through the production of IL-10. This suggests that the time course of burn wound healing may be influenced by these cells. In our experiments, CD31+ IMC are shown to suppress host antibacterial defense through the production of CCL2 and IL-10, and glycyrrhizin has improved host antibacterial resistance through the inhibition of CCL2 and IL-10 production by CD31+ IMC. To determine how wound healing is influenced by the glycyrrhizin treatment, further studies are required.
Resistance of γ-irradiated NOD-SCID IL-2rγ−/− mice treated with anti-AMP IgG to
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The authors wish to thanks the personnel of the Minophagen Pharmaceutical Co. Ltd., especially Drs. Tokuichiro Utsunomiya and Hideo Inoue for providing us glycyrrhizin, and Jihyang Chin and Yamato Okita for facilitating this study.