Antioxidants Protect Keratinocytes against M. ulcerans Mycolactone Cytotoxicity

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease

The human antimicrobial protein hCAP18/LL37 in wound healing and cell proliferation

Epithelia constitute the principal barriers separating the host and the potentially injurious environment, and defence of this interface is therefore essential. Antimicrobial peptides are key effector molecules of the innate immune system. hCAP18, the sole cathelicidin antimicrobial protein in humans, is present in leukocytes and is expressed in skin and other epithelia, indicative of a central role in barrier protection. The C-terminal peptide LL-37, enzymatically released from the holoprotein hCAP18, has the capacity to kill a wide range of pathogens as well as to modulate host cell responses. The aim of this thesis was to explore the role of LL-37 in barrier protection by investigating the expression of hCAP18/LL-37 in response to epithelial injury and, in addition, to investigate regulatory aspects of its expression. In this context we studied physiological wound healing in human skin in vivo and demonstrated significant upregulation of hCAP18/LL-37 upon wounding. The upregulation of hCAP18/LL-37 in the reepithelializing front following wounding in vivo was reproduced in a wound model of human skin ex vivo, indicating that this induction does not require inflammation. In contrast to the acute wounds, hCAP18/LL-37 levels in chronic ulcers were low and the protein was lacking in the wound edge epithelium. Inhibition studies employing anti-LL-37 serum to the ex vivo wounds hindered reepithelialization in a concentration-dependent manner. These results imply that LJ-37 is required for reepithelialization and that the deficiency of LL-37 in chronic ulcers may contribute to their failure to heal. Antimicrobial peptides have also been suggested to be involved in host defence against tumors. In this perspective we examined hCAP18/LL-37 expression in a number of breast carcinomas and discovered that hCAP18/LL-37 was distinctly expressed in tumor cells and not in the surrounding stroma. Not all tumor cells were positive for hCAP18/LL-37 and strongly positive cells were detected adjacent to cells devoid of hCAP18/LL-37, suggesting an intricate and tightly controlled expression of hCAP18/LL-37 in breast tumors. To investigate whether hCAP18/LL-37 would provide growth privilege for the tumor cells, human epithelial cells were treated with synthetic biologically active LL-37 peptide which resulted in significant increase in cell proliferation. Furthermore, transgenic overexpression of hCAP18 in human epithelial cells resulted in a significantly higher proliferation compared with control cells. These results do not support that hCAP18/LL37 acts as an anti-tumor agent, but rather suggest a role for hCAP18/LL-37 in promoting tumor growth. The molecular machinery commanding hCAP18/LL-37 gene expression is still unclear. With the purpose of investigating agents known to affect proliferation and differentiation of human skin, we treated human neonatal keratinocytes with vitamin D3 and biologically active analogues and observed marked upregulation of hCAP18 mRNA and protein syntheses. Pretreatment with calcium increased the hCAP18 expression and was synergistic with vitamin D3. Promoter studies revealed that vitamin D directly induced transcription of the hCAP18 gene by binding functional vitamin D-responsive elements in hCAP18 gene promoter. The effect was verified in vivo on intact human skin and in acute surgical wounds. These findings suggest that vitamin D is a key regulator of hCAP18/LL-37 in the skin. In conclusion, our results reveal novel properties of hCAP18/LL-37 demonstrating its critical participation in reepithelialization of human skin, its absence in chronic ulcers, its potential role in tumor growth and the discovery that vitamin D directly regulates hCAP18 gene expression in both intact and injured human skin in vivo

Substance P Antagonist Aprepitant Shows no Additive Effect Compared with Standardized Topical Treatment Alone in Patients with Atopic Dermatitis

Atopic dermatitis (AD) is a chronic, itchy, inflammatory skin disorder that may worsen due to stress and anxiety. Tachykinins have been suggested to be involved in the inflammation in AD, as well as pruritus. Aprepitant is a NK-1 receptor antagonist. This open randomized trial evaluated the effect of aprepitant added to topical treatment in adult patients with moderate-severe AD. The treatment group (n = 19) received 80 mg/day aprepitant for 7 days as a supplement to standardized topical treatment with a moderately strong steroid and a moisturizer. The control group (n = 20) received topical treatment alone. Patients were monitored for the extent of the disease (using SCORing of Atopic Dermatitis; SCORAD), pruritus, and scratching movements. In both the aprepitant-treated and the control groups there was a decrease in SCORAD, pruritus and scratching movements. How-ever, there was no significant additional improvement in any of these parameters in the aprepitant-treated group compared with the control group

Antioxidant protection against mycolactone cytotoxicity.

<p>(A) Antioxidants were added 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring WST-1color at 450 nm. Data represent means of triplicate determinations and standard deviations from one representative experiment out of two performed (*, p<0.05, one way ANOVA and students t-test). (B) Deferoxamine (D) and TEMPOL were added at different concentrations alone or in combination 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring neutral red uptake Concentrations are in µM for all substances except catalase which is in U/ml. Data shown are means and SEM of four experiments (*, p<0.05, ANOVA and students t-test). TEMPOL 1600 µM was not included in the statistical analysis (N = 2).</p

Mycolactone increases ROS production.

<p>Intracellular ROS production was detected by intracellular CM-H₂DCFDA fluorescence in keratinocytes after 45 min incubation in control medium or with 100 and 300 ng/ml mycolactone. Parallel cultures were pretreated for 30 min with a combination of deferoxamine (100 µM) and TEMPOL (200 µM) (D+T) before CM-H₂DCFDA and addition of mycolactone. Asterisks indicate significant (p<0.05) differences in fluorescence between treatments and their corresponding control in one representative experiment with four to six replicates (means and SD).</p

Mycolactone is cytotoxic for keratinocytes.

<p>Mycolactone was added to sub-confluent cultures of keratinocytes and (A) cell numbers were measured as optical density of WST-1after 24, 48 and 72 h of treatment with 1000 ng/ml of mycolactone, or (B) after 72 h at different concentrations of mycolactone. An asterisk indicates a significant (p<0.05) reduction in WST-1 labeling as compared to the corresponding medium control. Cell cultures of untreated controls (C) and cultures treated with 300 ng/m mycolactone (D) were photographed after 48 h. Data are from one representative experiment showing means and SD of triplicates.</p