AMPs are an integral part of the innate immune response as they have been shown to be effective in killing bacterial3
pathogens. Cathelicidin is produced by several cells in the skin including keratinocytes where it is induced in response to inflammatory stimuli.12,13
Upon release, the cathelicidin precursor protein is processed into the biologically active antimicrobial peptide LL-37. In this study we demonstrate that LL-37 exhibits anti-viral activity against HSV. Our previous studies have demonstrated that AD patients, in general, have a reduced ability to generate cathelicidin in their skin, as compared to patients with psoriasis or allergic contact dermatitis, and this may predispose them to microbial skin infection.3,14
However, the propensity of AD patients to serious skin infection such as ADEH has not previously been explored. Results from the current study indicate that a more exaggerated reduction in cathelicidin expression may predispose a subset of AD patients to develop ADEH. Thus, there is heterogeneity in the expression of cathelicidin within AD such that the individuals with the lowest levels are most prone to disseminated viral infection.
It was previously reported that LL-37 exhibits little activity against HSV-1 or -2.15
In contrast, we demonstrate in this study that LL-37 exhibits potent anti-viral activity against HSV. In the previous study, Yasin et al.
demonstrated that 44.5 μM of LL-37 provided 28 and 46% protection against HSV-1 and HSV-2, respectively, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. In the MTT assay, anti-viral activity of LL-37 is determined by increases in cellular proliferation compared to cells treated with virus alone. In our current study, we measured HSV replication by evaluating glycoprotein D gene expression using real-time RT-PCR. By directly measuring the virus activity rather than cellular proliferation, it provides a more direct and appropriate measurement of the anti-viral activity of LL-37 against HSV accounting for differences between our results and those of Yasin et al.15
Our observation is further supported by Gordon et al.16
who recently demonstrated that LL-37 exhibits anti-viral activity against HSV-1 in corneal and conjunctival epithelia.
In this study, we demonstrate that concentrations as low as 10 μM of LL-37 reduce HSV levels by over 10,000-fold. Since psoriatic skin can contain up to 1605 micromolar LL-37,3
this demonstrates that physiologic concentrations of LL-37 are effective at controlling HSV replication. This was further supported using a more physiological approach in which keratinocytes were pre-infected with HSV for six hours and then incubated with LL-37 for an additional 18 hours. Again, we saw greater than a 60% reduction in the levels of HSV when as little as 25 μM of LL-37 was added to previously infected keratinocytes. The importance of cathelicidins in skin innate immune responses to HSV is also strongly supported by our current finding that skin explants from mice deficient in the cathelicidin gene Cnlp, and it's AMP product CRAMP, sustain higher levels of HSV replication following inoculation as compared to their wild type counterparts. Mouse CRAMP is very similar to human LL-37 in structure, tissue distribution and antimicrobial activity and is therefore a reasonable model of the human cathelicidin. The observation that CRAMP deficient mice support a higher level of HSV replication reinforces the important impact that a cathelicidin deficiency would have on HSV skin infection in humans. These data support a conclusion that decreased cathelicidin expression will significantly increase the potential for disseminated skin infection to occur.
Since not all AD patients develop ADEH, we investigated the abundance of cathelicidin in the skin of AD patients and ADEH patients to determine whether the development of ADEH corresponded with decreased cathelicidin expression. Skin biopsies were obtained from naturally induced inflammatory skin rashes of AD and ADEH. This allowed for comparisons between similarly stimulated skin samples. Lesional skin from ADEH patients exhibited significantly lower levels of cathelicidin protein than skin lesions of uncomplicated AD patients. Our results suggest that AD patients with the lowest levels of cathelicidin are most susceptible to developing ADEH and that lack of this molecule may serve as a biomarker for patients at risk for disseminated viral skin infection.
AD skin is characterized by the over-expression of the Th2 cytokines IL-4 and IL-13.16
Previously we demonstrated that Th2 cytokines down-regulate cathelicidin.10
However, due to the difficulty in identifying ADEH patients and insufficient amounts of archived tissue, we were unable to investigate the levels of IL-4 and IL-13 in AD and ADEH patients for potential differences. IL-4 and IL-13 are Th2 cytokines essential in the production of IgE.10
Therefore, the measurement of serum IgE may serve as a biomarker for levels of Th2 responses. Wollenberg et al.6
and Lagace-Simard et al.18
have previously demonstrated that ADEH patients exhibited higher total serum IgE levels than AD patients. We confirmed this observation in the current study by demonstrating significantly higher serum IgE levels in AD patients as compared to ADEH patients. We further determined that there is a strong correlation between the levels of serum IgE and cathelicidin protein expression in AD and ADEH patients. Therefore serum IgE levels may serve as a surrogate marker for the expression of cathelicidin in the skin of AD and ADEH patients and may separate those who are more susceptible to disseminated viral infection.
The current study therefore demonstrates that AD is a heterogeneous population of patients expressing different levels of cathelicidin in the skin. This explains, in part, why a subgroup of AD patients is susceptible to developing ADEH following HSV infection. Additionally this study demonstrates the importance of the cathelicidin in controlling the replication of HSV in the skin. This is supported by significantly higher levels of HSV replication in the skin of Cnlp knockout mice and the significant reduction of HSV gene expression in keratinocytes treated with LL-37. Overall these data suggest further clinical studies are warranted to examine whether augmentation of LL-37 expression in AD skin may be useful in prevention of ADEH and the perplexing challenge of controlling microbial infection in this common skin problem.