Cathelicidin deficiency predisposes to eczema herpeticum

Abstract

Background: The cathelicidin family of antimicrobial peptides is an integral component of the innate immune response that exhibits activity against bacterial, fungal, and viral pathogens. Eczema herpeticum (ADEH) develops in a subset of patients with atopic dermatitis (AD) because of disseminated infection with herpes simplex virus (HSV).

Objective: This study investigated the potential role of cathelicidins in host susceptibility to HSV infection.

Methods: Glycoprotein D was measured by means of real-time RT-PCR as a marker of HSV replication in skin biopsy specimens and human keratinocyte cultures. Cathelicidin expression was evaluated in skin biopsy specimens from patients with AD (n = 10) without a history of HSV skin infection and from patients with ADEH (n = 10).

Results: The cathelicidin peptide LL-37 (human cathelicidin) exhibited activity against HSV in an antiviral assay, with significant killing (P < .001) within the physiologic range. The importance of cathelicidins in antiviral skin host defense was confirmed by the observation of higher levels of HSV-2 replication in cathelicidin-deficient (Cnlp-/-) mouse skin (2.6 +/- 0.5 pg HSV/pg GAPDH, P < .05) compared with that seen in skin from their wild-type counterparts (0.9 +/- 0.3). Skin from patients with ADEH exhibited significantly (P < .05) lower levels of cathelicidin protein expression than skin from patients with AD. We also found a significant inverse correlation between cathelicidin expression and serum IgE levels (r2 = 0.46, P < .05) in patients with AD and patients with ADEH.

Conclusion: This study demonstrates that the cathelicidin peptide LL-37 possesses antiviral activity against HSV and demonstrates the importance of variable skin expression of cathelicidins in controlling susceptibility to ADEH. Additionally, serum IgE levels might be a surrogate marker for innate immune function and serve as a biomarker for which patients with AD are susceptible to ADEH.

Clinical implications: A deficiency of LL-37 might render patients with AD susceptible to ADEH. Therefore increasing production of skin LL-37 might prevent herpes infection in patients with AD.

Figure 1

LL-37 exhibits anti-viral activity against HSV. Physiologic concentrations of LL-37 were pre-incubated with HSV-2 for 24 hours and then added to BS-C-1 for an additional 24 hours to evaluate HSV-2 gene expression by real-time RT-PCR (A) or 48 hours to investigate functionally active virus using a standard plaque assay (B). *** indicates a significant difference of p<0.001 as compared to 0 μM.

Figure 1

LL-37 exhibits anti-viral activity against HSV. Physiologic concentrations of LL-37 were pre-incubated with HSV-2 for 24 hours and then added to BS-C-1 for an additional 24 hours to evaluate HSV-2 gene expression by real-time RT-PCR (A) or 48 hours to investigate functionally active virus using a standard plaque assay (B). *** indicates a significant difference of p<0.001 as compared to 0 μM.

Figure 2

Exogenous LL-37 rescues HSV infected keratinocytes. Human keratinocytes were infected with 0.05 pfu/cell HSV-2 for six hours and then treated with physiologic concentrations of LL-37 for an additional 18 hours. RNA was isolated from the cells and the levels of HSV-2 gene expression evaluated by real-time RT-PCR. **indicates a significant difference of p<0.01 as compared to HSV alone.

Figure 3

Essential role of cathelicidins in controlling HSV replication in the skin. Skin biopsies from BALB/c (n=5) and Cnlp KO (n=5) mice were stimulated with HSV-2 for 24 hours and evaluated for HSV-2 gene expression. RNA was collected from the tissue and the levels of HSV-2 evaluated by real-time RT-PCR. *indicates a significant difference of p<0.05.

Figure 4

Expression of cathelicidin elevated in AD as compared to ADEH skin. A. Paraffin embedded skin explants from AD (n=10) and ADEH (n=10) patients were cut into 5 μm sections and stained for human cathelicidin. B. The intensity of the immunostaining was visually scored on a scale from 0 to 5, with 0 indicating no staining and 5 the most intense staining. *indicates statistical significance of p<0.05.

Figure 4

Expression of cathelicidin elevated in AD as compared to ADEH skin. A. Paraffin embedded skin explants from AD (n=10) and ADEH (n=10) patients were cut into 5 μm sections and stained for human cathelicidin. B. The intensity of the immunostaining was visually scored on a scale from 0 to 5, with 0 indicating no staining and 5 the most intense staining. *indicates statistical significance of p<0.05.

Figure 5

Correlation between serum IgE levels and cathelicidin expression in AD and ADEH patients. Serum IgE levels were determined from AD (n=9) and ADEH (n=9) patients. Regression analysis was performed on log transformed serum IgE values and LL-37 protein expression.