Paul Kotol

Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis

Commensal skin bacteria produce previously unknown antimicrobial peptides that can inhibit Staphylococcus aureus colonization of atopic dermatitis subjects. Bacterial biological warfare in atopic dermatitis Normal human skin is colonized by a variety of bacteria, which typically do not perturb the host. However, Staphylococcus aureus is known to aggravate symptoms of atopic dermatitis. Nakatsuji et al. report that other strains of Staphylococcus residing on the skin of healthy individuals produce a novel antimicrobial peptide that can inhibit S. aureus growth. Colonization of pigskin or mice with these protective commensals reduced S. aureus replication. Autologous bacterial transplant in a small number of atopic dermatitis patients drastically reduced S. aureus skin burden. This commensal skin transplant is already approved by the U.S. Food and Drug Administration, with a clinical trial underway. The microbiome can promote or disrupt human health by influencing both adaptive and innate immune functions. We tested whether bacteria that normally reside on human skin participate in host defense by killing Staphylococcus aureus, a pathogen commonly found in patients with atopic dermatitis (AD) and an important factor that exacerbates this disease. High-throughput screening for antimicrobial activity against S. aureus was performed on isolates of coagulase-negative Staphylococcus (CoNS) collected from the skin of healthy and AD subjects. CoNS strains with antimicrobial activity were common on the normal population but rare on AD subjects. A low frequency of strains with antimicrobial activity correlated with colonization by S. aureus. The antimicrobial activity was identified as previously unknown antimicrobial peptides (AMPs) produced by CoNS species including Staphylococcus epidermidis and Staphylococcus hominis. These AMPs were strain-specific, highly potent, selectively killed S. aureus, and synergized with the human AMP LL-37. Application of these CoNS strains to mice confirmed their defense function in vivo relative to application of nonactive strains. Strikingly, reintroduction of antimicrobial CoNS strains to human subjects with AD decreased colonization by S. aureus. These findings show how commensal skin bacteria protect against pathogens and demonstrate how dysbiosis of the skin microbiome can lead to disease.

Cathelicidin antimicrobial peptide LL-37 in psoriasis enables keratinocyte reactivity against TLR9 ligands

Here we show that keratinocytes in psoriatic lesional skin express increased Toll-like receptor (TLR) 9 that similarly localizes with elevated expression of the cathelicidin antimicrobial peptide LL-37. In culture, normal human keratinocytes exposed to LL-37 increased TLR9 expression. Furthermore, when keratinocytes were exposed to LL-37 and subsequently treated with TLR9 ligands such as CpG or genomic DNA, keratinocytes greatly increased production of type I interferons. This response mimicked observations in the epidermis of psoriatic lesional skin as keratinocytes in psoriatic lesions produce greater amounts of interferon-β than normal skin lacking LL-37. The mechanism for induction of type I interferons in keratinocytes was dependent on TLR9 expression but not on a DNA-LL-37 complex. These findings suggest that keratinocytes recognize and respond to DNA and can actively participate in contributing to the immunological environment that characterizes psoriasis.

The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

A randomized controlled double-blind investigation of the effects of vitamin D dietary supplementation in subjects with atopic dermatitis

Subjects with atopic dermatitis (AD) have defects in antimicrobial peptide (AMP) production possibly contributing to an increased risk of infections. In laboratory models, vitamin D can alter innate immunity by increasing AMP production.

The Host Defense Peptide Cathelicidin Is Required for NK Cell-Mediated Suppression of Tumor Growth

Tumor surveillance requires the interaction of multiple molecules and cells that participate in innate and the adaptive immunity. Cathelicidin was initially identified as an antimicrobial peptide, although it is now clear that it fulfills a variety of immune functions beyond microbial killing. Recent data have suggested contrasting roles for cathelicidin in tumor development. Because its role in tumor surveillance is not well understood, we investigated the requirement of cathelicidin in controlling transplantable tumors in mice. Cathelicidin was observed to be abundant in tumor-infiltrating NK1.1+ cells in mice. The importance of this finding was demonstrated by the fact that cathelicidin knockout mice (Camp −/−) permitted faster tumor growth than wild type controls in two different xenograft tumor mouse models (B16.F10 and RMA-S). Functional in vitro analyses found that NK cells derived from Camp −/− versus wild type mice showed impaired cytotoxic activity toward tumor targets. These findings could not be solely attributed to an observed perforin deficiency in freshly isolated Camp −/− NK cells, because this deficiency could be partially restored by IL-2 treatment, whereas cytotoxic activity was still defective in IL-2-activated Camp −/− NK cells. Thus, we demonstrate a previously unrecognized role of cathelicidin in NK cell antitumor function.

History of eczema herpeticum is associated with the inability to induce human β-defensin (HBD)-2, HBD-3 and cathelicidin in the skin of patients with atopic dermatitis

MADAM, Patients with atopic dermatitis (AD) with a history of eczema herpeticum (ADEH) represent a subset of individuals with AD who have more severe disease.1 It has previously been shown that the skin of patients with AD has defects in its capacity to increase maximally the antimicrobial peptides (AMPs), cathelicidin, human β-defensin (HBD)-2 and HBD-3 in response to inflammation, in contrast to patients with psoriasis who also have inflammation of the skin, but no difficulty with AMP induction.2,3 To date, however, previous studies have not examined HBD-2 and HBD-3 induction in the ADEH subset of patients.4 We sought to examine if subjects with ADEH exhibit an inhibition in induction of AMPs, compared with subjects without ADEH. A total of 81 subjects were recruited into the study: 26 patients with psoriasis, 21 with AD and nine with ADEH and 25 healthy subjects with no history of skin disease (Table S1, see Supporting information). Two 2-mm punch biopsies were collected from lesional and nonlesional skin of the patients. Normal controls provided two 2-mm punch biopsies from the skin of their upper inner arm. Quantification of cathelicidin, interleukin (IL)-13, HBD-2 and HBD-3 in the skin was performed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) (see supporting information), and their expression was calculated as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. All statistical analyses were performed using SAS v9.1 or higher (SAS Institute, Cary, NC, U.S.A.) (see Supporting information). Compared with individuals with AD or psoriasis, patients with ADEH showed a lesser relative increase in cathelicidin, HBD-2 and HBD-3 mRNA in their lesional skin (Fig. 1). Immunohistochemical staining of LL-37 confirmed the RT-PCR data for this peptide product of the cathelicidin gene (Fig. S1, see Supporting information) and supported a prior report that cathelicidin is suppressed in subjects with ADEH.4 This finding, combined with the novel observation of decreased inducibility of HBD-2 and HBD-3, suggests that HBD-2 and HBD-3 may further contribute to the increased incidence of secondary infection with herpes simplex virus (HSV) in ADEH. Fig 1 (a)Ability to induce antimicrobial peptides (AMPs) as evidenced by lesional minus nonlesional expression analysed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Skin biopsy samples from lesional and nonlesional skin ... As expected, due to low expression in normal skin, nonlesional skin revealed no differences in cathelicidin levels between the four groups (Fig. 2).5 Interestingly, however, nonlesional HBD-2 levels were significantly higher in patients with ADEH compared with those with AD (P < 0.001) or psoriasis (P < 0.001) and normal controls (P = 0.001). Nonlesional HBD-3 levels were also higher in patients with ADEH compared with those with AD (P = 0.002) or psoriasis (P = 0.003). Upregulation of HBD-2 and HBD-3 has been shown to occur with proinflammatory stimuli such as tumour necrosis factor (TNF)-α and interferon (IFN)-γ through signal transducer and activator of transcription (STAT)-1 and nuclear factor-κB, and inhibition through IL-4 and IL-13 to activate STAT-6 to induce the suppressors of cytokine signalling (SOCS)-1 and SOCS-3 which inhibit TNF-α and IFN-γ.6 The presence of nonpathogenic organisms such as Staphylococcus epidermidis may be the signal for the induction of TNF-α or IFN-γ, which may outweigh the downregulatory effects of IL-13 and IL-4. Fig 2 (a) Nonlesional skin values for cathelicidin analysed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) revealed no significant differences ... Significantly higher serum IgE levels were observed in subjects with ADEH than in patients with AD (P < 0.001) or controls (P < 0.001), suggesting that the T-helper (Th) 2 phenotype is more prominent in individuals with ADEH compared with patients with AD. Examination of lesional minus nonlesional skin cathelicidin levels for patients with ADEH and AD combined revealed a significant negative correlation with total serum IgE (r = −0.602, P = 0.018), again indicating that increased Th2 polarity reduces the ability of the atopic skin to induce cathelicidin during inflammation (Fig. S2, see Supporting information). Finally, as IL-13 has been associated with the Th2 phenotype, IL-13 levels were analysed to determine if the cytokine milieu differed in lesional and nonlesional skin. Lesional IL-13 levels were significantly higher in subjects with ADEH than in patients with AD (P = 0.018) or psoriasis (P < 0.001). Nonlesional IL-13 levels also presented a similar trend, with levels in ADEH nonlesional skin significantly higher than in the nonlesional skin of patients with AD (P = 0.005) or psoriasis (P = 0.014). As expected, lesional IL-13 levels of subjects with ADEH and AD combined showed a positive correlation with total IgE (r = 0.699, P = 0.017) (Fig. S3, see Supporting information). In summary, our study shows that there is a defect in the inducibility of the AMPs, HBD-2, HBD-3 and cathelicidin, in patients with ADEH. This indicates that individuals with ADEH are deficient in several AMPs, which may account for their increased risk of infections with Staphylococcus aureus and HSV as well as their propensity for eczema vaccinatum. Nonlesional skin values for HBD-2 and HBD-3 are not depressed in patients with ADEH compared with those with AD, normal controls or patients with psoriasis. These observations suggest that analysis of AMP expression may be a useful tool for the prediction of superinfection in individuals with AD; however future studies with larger numbers of patients may be necessary to confirm this finding. Furthermore, understanding the factors that contribute to the suppression of AMP expression in AD can help inform the design of novel strategies for correcting this defect.

Bcl-3 Acts as an Innate Immune Modulator by Controlling Antimicrobial Responses in Keratinocytes

Innate immune responses involve the production of antimicrobial peptides (AMPs), chemokines, and cytokines. We report here the identification of B-cell leukemia (Bcl)-3 as a modulator of innate immune signaling in keratinocytes. In this study, it is shown that Bcl-3 is inducible by the Th2 cytokines IL-4 and IL-13 and is overexpressed in lesional skin of atopic dermatitis (AD) patients. Bcl-3 was shown to be important to cutaneous innate immune responses as silencing of Bcl-3 by small-interfering RNA (siRNA) reversed the downregulatory effect of IL-4 on the HBD3 expression. Bcl-3 silencing enhanced vitamin D3 (1,25D3)-induced gene expression of cathelicidin AMP in keratinocytes, suggesting a negative regulatory function on cathelicidin transcription. Furthermore, 1,25D3 suppressed Bcl-3 expression in vitro and in vivo. This study identified Bcl-3 as an important modulator of cutaneous innate immune responses and its possible therapeutic role in AD.

Reduction in serine protease activity correlates with improved rosacea severity in a small, randomized pilot study of a topical serine protease inhibitor.

Individuals with rosacea express high baseline levels of cathelicidin and kallikrein 5 (KLK5) (Yamasaki, et al, 2007). The significance of elevated KLK5 is that this trypsin-like serine protease controls the cleavage of the cathelicidin precursor protein into LL-37 (Yamasaki et al, 2007; Yamasaki et al, 2006). LL-37 is both pro-inflammatory and angiogenic (Koczulla et al, 2003; Lande et al, 2004; Morizane et al, 2012). Based on these findings, we hypothesized that inhibition of KLK5 may improve the clinical signs of rosacea by decreasing LL-37 production.

The Cutaneous Microbiome and Aspects of Skin Antimicrobial Defense System Resist Acute Treatment with Topical Skin Cleansers

The human skin microbiome has been suggested to play an essential role in maintaining health by contributing to innate defense of the skin. These observations have inspired speculation that the use of common skin washing techniques may be detrimental to the epidermal antibacterial defense system by altering the microbiome. In this study, several common skin cleansers were used to wash human forearms and the short-term effect on the abundance of the antimicrobial peptide LL-37 and the abundance and diversity of bacterial DNA was measured. Despite small but significant decreases in the amount of LL-37 on the skin surface shortly after washing, no significant change in the bacterial community was detected. Furthermore, Group A Streptococcus did not survive better on the skin after washing. In contrast, the addition of antimicrobial compounds such as benzalkonium chloride or triclocarban to soap before washing decreased the growth of Group A Streptococcus applied after rinse. These results support prior studies that hand washing techniques in the health care setting are beneficial and should be continued. Additional research is necessary to better understand the effects of chronic washing and the potential impact of skin care products on the development of dysbiosis in some individuals.

Etanercept decreases the innate immune wounding response in psoriasis.

Cathelicidin is increased when normal skin is injured and in psoriasis lesions where it has been suggested to play a pivotal role in inflammation through interactions with self‐DNA and toll‐like receptor 9 (TLR‐9) in keratinocytes and plasmacytoid dendritic cells. Because of etanercepts success in treating psoriasis, we hypothesized that etanercept may suppress TLR‐9 and cathelicidin induction. Examination of experimentally induced wounds of psoriatic lesional and non‐lesional skin, and comparison with wounded normal skin, shows that the induction of cathelicidin and TLR‐9 is greatly enhanced in lesional psoriatic skin. Six weeks of etanercept appears not to affect the baseline expression of cathelicidin or TLR‐9, but does blunt the induction of cathelicidin in psoriasis with wounding. These findings support the role of cathelicidin in the enhancement of local inflammation in psoriasis and may partially explain one of the mechanisms enabling TNF‐α inhibitors to successfully treat this disorder.