A Role of Staphyococcus aureus, Interleukin-18, Nerve Growth Factor and Semaphorin 3A, an Axon Guidance Molecule, in Pathogenesis and Treatment of Atopic Dermatitis

Abstract

Staphylococcus aureus (SA) is usually present not only in the skin lesions of atopic dermatitis (AD) but also in the atopic dry skin. SA discharges various toxins and enzymes that injure the skin, results in activation of epidermal keratinocytes, which produce and release IL-18. IL-18 that induces the super Th1 cells secreting IFN-γ and IL-13 is supposed to be involved in development of AD and its pathogenesis. Indeed, the number of SA colonies on the skin surface and the serum IL-18 levels in patients with AD significantly correlated with the skin scores of AD lesions. Also, there is strong positive correlation between the skin scores and serum IL-18 levels in DS-Nh mice (P<0.0001, r=0.64), which develop considerable AD-like legions when they are housed under conventional conditions, but develop skin legions with less severity and less frequency under specific pathogens free (SPF) conditions. Therefore, they are well-known as model mice of AD, in which SA is presumed to be critical factor for the development of AD lesions. Also, theses DS-Nh mice pretreated with Cy developed more remarkable AD-like lesions in comparison with non-treated ones. The levels of INF-r and IL-13 in the supernatants of the lymph node cell cultures stimulated with staphylococcal enterotoxin B (SEB) or ConA were increased in the Cy-treated mice, although the serum levels of total IgE were not. In this experiment, we revealed that Cy-treated mice, to which CD25 +CD4 + reguratory T cells taken from non-treated ones had been transferred, developed the AD-like legions with less severity and less number of SA colonies on the skin surface. Therefore, it is presumed that CD25 +CD4 + reguratory T cells might be involved in the suppression of super Th1 cells which are induced by IL-18 and are involved in the development of AD-like lesions rather than IgE production. The efficient induction of CD25 +CD4 + reguratory T cells is expected for the new type of treatment of AD. We also found that farnesol (F) and xylitol (X) synergistically inhibited biofilm formation by SA, and indeed the ratio of SA in total bacteria at sites to which the FX cream containing F and X had been applied was significantly decreased 1 week later, accompanied with improvement of AD, when compared with that before application and at placebo sites. Therefore, the FX cream is a useful skin-care agent for atopic dry skin colonized by SA. The nerve growth factor (NGF) in the horny layer (the horn NGF) of skin lesions on the cubital fossa was collected by tape stripping and measured using ELISA in AD patients before and after 2 and 4 weeks treatments. Simultaneously, the itch and eruptions on the whole body and on the lesions, in which the horn NGF was measured, were recorded, and also the peripheral blood eosinophil count, serum LDH level and serum total IgE level were examined. The level of NGF was significantly higher in AD patients than in healthy controls, correlated with the severity of itch, erythema, scale/xerosis, the eosinophil count and LDH level, and also significantly decreased after treatments with olopatadine and/or steroid ointment for 2 and 4 weeks. Therefore, the measurement of the NGF by this harmless method seems to be useful to assess the severity of AD and the therapeutic effects on AD. In AD patients, C-fiber in the epidermis increase and sprout, inducing hypersensitivity, which is considered to aggravate the disease. Semaphorin 3A (Sema3A), an axon guidance molecule, is a potent inhibitor of neurite outgrowth of sensory neurons. We administered recombinant Sema3A intracutaneously into the skin lesions of NC/Nga mice, an animal model of AD, and investigated the effect of Sema3A on the skin lesions and their itch. Sema3A dose-dependently improved skin lesions and attenuated the scratching behavior in NC/Nga mice. Histological examinations revealed a decrease in the epidermal thickness, the density of invasive nerve fibers in the epidermis, inflammatory infiltrate including mast cells and CD4 +T cells, and the production of IL-4 in the Sema3A-treated lesions. Because the interruption of the itch-scratch cycle likely contributes to the improvement of the AD-like lesions, Sema3A is expected to become a promising treatment of patients with refractory AD.

Keywords: Atopic dermatitis; Semaphorin 3A; Staphylococcus aureus; nerve growth factor.

Fig. 1

The number of SA detected on the skin surface of forearm and forehead in patients with AD is remarkably more than in healthy controls. cfu: colony forming unit.

Fig. 2

The correlation between eruption score and the number of SA on the same area of patients with AD. cfu: colony forming unit.

Fig. 3-1

Farnesol and xylitol (FX) have an inhibitory effect on biofilm formation of SA. A biofilm of bacteria was formed by incubating a plastic coverslip coated with type IV collagen in each well of a 24-well tissue culture plate with the human plasma and TSB (1:1) medium, into which SA was inoculated. Cell suspensions of SA from an AD patient (1×10⁸ cfu/mL) were inoculated separately into 1 mL of the medium either alone (control) or supplemented with FX. After incubation at 37℃, each coverslip was observed visually and also under a scanning electron microscope (SEM) and number of SA colony adhered to the coverslip of was counted. TSB: tryptic soy broth.

Fig. 3-2

FX dissolve the formed biofilm of SA.

Fig. 4

The treatment with FX cream improved significantly 4 items out of 5 items in AD lesions 2 weeks later, while the treatment with control cream improved only 1 item of dryness/desquamation.

Fig. 5

Application of FX cream containing 0.2% farnesol and 5% xylitol to AD patients for 2 weeks decreased the number and rate of SA significantly when compared with the number at the start and with placebo control treatment. FX: cream containing 0.2% farnesol and 5% xylitol, CT: control treatment, ^#the rate (%) of SA to total bacteria, N.S. : not significant, ^**P < 0.05, ^***P < 0.01.

Fig. 6-1

Comparison between serum levels of IL-18 and global skin scores in AD patients divided into 4 groups based on their disease severities.

Fig. 6-2

Comparison between serum IL-18 levels and skin scores in DS-Nh mice (n=14 each group).

Fig. 7

FoxP3⁺CD25⁺CD4⁺ regulatory T cells are presumed to negatively regulate the induction and exercerbation of AD-like lesions through the suppression of IFNγ/L-13 production of by FoxP3^-CD25^-CD4⁺ super Th1 cells in DS-Nh of AD model mouse associated with SA colonization on the skin surface.,

Fig. 8

NGF levels in the horny proteins.

Fig. 9

Effect of treatment on horny NGF of AD patients.

Fig. 10

Semaphorin family and its receptorsSema., D: Semadomain, Ig: immunoglobulin, BD: Basic domain, TSP: Thrombospondin repeat, GPI: Glycerophosphatidyl-inositol anchor, MRS: Met-related sequence, G-P: glycine proline repeat, CUB: complement binding domains (a1 and a2 domains), FV/VIII: FactorV/VIII coagulation factor-like domains (b1 and b2 domains), MAM: Mepulin-A5-µ domain.

Fig. 11-1

Sema3A improves AD-like lesions of NC/Nga, AD model mouse. The clinical skin score was defined as the sum of individual scores graded as 0 (none), 1 (mild), 2 (moderate) and 3 (severe) for the symptoms of erythema/hemorrhage, edema, excoriation/erosion and scaling/dryness. ^*P < 0.05, ^**P < 0.01, compared to no treatment group and control group.

Fig. 11-2

Sema3A improves clinical skin score in a dose-dependent manner. The clinical skin score was defined as the sum of individual scores graded as 0 (none), 1 (mild), 2 (moderate) and 3 (severe) for the symptoms of erythema/hemorrhage, edema, excoriation/erosion and scaling/dryness. ^*P < 0.05, ^**P < 0.01, compared to no treatment group and control group.

Fig. 11-3

Sema3A suppresses scratching behavior of NC/Nga mice.

Fig. 12

Pathomechanism of itch and action mechanism of Sema3A on itching of AD lesions: Sema3A has not only the neurological activity, which inhibits the intraepidermal extension of peripheral nerve, but also the immunological anti-inflammatory activity, and is expected as a new type of drug effective for refractory AD patients who are resistant to existing drugs.