The Transcription Factor p63 Is a Direct Effector of IL-4- and IL-13-Mediated Repression of Keratinocyte Differentiation.

Atopic Dermatitis is an inflammatory skin disease associated with broad defects in skin barrier function caused by increased levels of type-2 cytokines (IL-4 and IL-13) that repress keratinocyte (KC) differentiation. Although crucial in mediating allergic disease, the mechanisms for gene repression induced by type-2 cytokines remain unclear. In this study, we determined that gene repression requires the master regulator of the epidermal differentiation program, p63. We found that type-2 cytokine-mediated inhibition of the expression of genes involved in early KC differentiation, including keratin 1, keratin 10, and DSC-1, is reversed by p63 blockade. Type-2 cytokines, through p63, also regulate additional genes involved in KC differentiation, including CHAC-1, STC2, and CALML5. The regulation of the expression of these genes is ablated by p63 small interfering RNA as well. In addition, we found that IL-4 and IL-13 and Staphylococcus aureus lipoteichoic acid work in combination through p63 to further suppress the early KC differentiation program. Finally, we found that IL-4 and IL-13 also inhibit the activity of Notch, a transcription factor required to induce early KC differentiation. In conclusion, type-2 cytokine-mediated gene repression and blockade of KC differentiation are multifactorial, involving pathways that converge on transcription factors critical for epidermal development, p63 and Notch.

Staphylococcus aureus Lipoteichoic Acid Damages the Skin Barrier through an IL-1-Mediated Pathway.

Staphylococcus aureus is a significant bacterial pathogen that may penetrate through the barrier into the epidermis and dermis of the skin. We hypothesized that the S. aureus cell wall product lipoteichoic acid (LTA) may contribute to the development of inflammation and skin barrier defects; however, the effects of LTA in vivo are not well understood. In this study, we examined the effects induced by intradermal S. aureus LTA. We found that keratinocytes in LTA-treated skin were highly proliferative, expressing 10-fold increased levels of Ki67. Furthermore, we observed that LTA caused damage to the skin barrier with substantial loss of filaggrin and loricrin expression. In addition, levels of the IL-1 family of inflammatory cytokines, as well as the neutrophil-attracting chemokines Cxcl1 and Cxcl2, were increased. Concomitantly, we observed significant numbers of neutrophils infiltrating into the epidermis. Finally, we determined that LTA-induced signals were mediated in part through IL-1, because an IL-1 receptor type 1 antagonist ameliorated the effects of LTA, blocking neutrophil recruitment and increasing the expression of skin barrier proteins. In summary, we show that S. aureus LTA alone is sufficient to promote keratinocyte proliferation, inhibit expression of epidermal barrier proteins, induce IL-1 signaling, and recruit cells involved in skin inflammation.

Skin Wound Healing Is Accelerated by a Lipid Mixture Representing Major Lipid Components of Chamaecyparis obtusa Plant Extract.

In chronic nonhealing wounds, the healing process is disrupted and wounds are often infected with bacteria. About 85% of lower extremity amputations in diabetes are attributed to deep infection of foot ulcers. Therefore, infection control is critical for wound care. In this study, we analyzed lipid composition of Chamaecyparis obtusa extract, and we describe the wound-healing properties of its combination of 10 major lipid components. A 10-lipid mixture up-regulated HBD-3 and LL-37 through the olfactory receptor 2AT4 and induced phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases in primary human keratinocytes. In addition, the 10-lipid mixture had direct bactericidal effects against Staphylococcus aureus and Streptococcus pyogenes and protected against staphylococcal α-toxin-induced keratinocyte cell death. In an animal model, the 10-lipid mixture accelerated skin wound healing and was also effective in healing wounds superinfected with S. aureus. We suggest that the 10-lipid mixture, because of its wound-healing and antimicrobial properties, can be beneficial for wound treatment.

Interferon-γ Protects from Staphylococcal Alpha Toxin-Induced Keratinocyte Death through Apolipoprotein L1.

Staphylococcus aureus is a bacterial pathogen that frequently infects the skin, causing lesions and cell destruction through its primary virulence factor, alpha toxin. Here we show that interferon gamma (IFN-?) protects human keratinocytes from cell death induced by staphylococcal alpha toxin. We find that IFN-? prevents alpha toxin binding and reduces expression of the alpha toxin receptor, a disintegrin and metalloproteinase 10 (ADAM10). We determine that the mechanism for IFN-?-mediated resistance to alpha toxin involves the induction of autophagy, a process of cellular adaptation to sublethal damage. We find that IFN-? potently stimulates activation of the primary autophagy effector, light chain 3 (LC3). This process is dependent on upregulation of apolipoprotein L1. Depletion of apolipoprotein L1 by small interfering RNA significantly increases alpha toxin-induced lethality and inhibits activation of light chain 3. We conclude that IFN-? plays a significant role in protecting human keratinocytes from the lethal effects of staphylococcal alpha toxin through apolipoprotein L1-induced autophagy.

Th2 cytokines increase Staphylococcus aureus alpha toxin-induced keratinocyte death through the signal transducer and activator of transcription 6 (STAT6).

Atopic dermatitis (AD) is an inflammatory skin disease characterized by increased T-helper type 2 (Th2) cytokine expression. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the lytic virulence factor, alpha toxin. In the current study, we report that alpha toxin-induced cell death is greater in the skin from patients with AD compared with controls. Furthermore, we find that keratinocyte differentiation and Th2 cytokine exposure influence sensitivity to S. aureus alpha toxin-induced cell death. Differentiated keratinocytes are protected from cell death, whereas cells treated with Th2 cytokines have increased sensitivity to alpha toxin-induced lethality. Our data demonstrate that the downstream effects mediated by Th2 cytokines are dependent upon host expression of STAT6. We determine that Th2 cytokines induce biochemical changes that decrease levels of acid sphingomyelinase (SMase), an enzyme that cleaves sphingomyelin, an alpha toxin receptor. Furthermore, Th2 cytokines inhibit the production of lamellar bodies, organelles critical for epidermal barrier formation. Finally, we determine that SMase and its enzymatic product, phosphocholine, prevent Th2-mediated increases in alpha toxin-induced cell death. Therefore, our studies may help explain the increased propensity for Th2 cytokines to exacerbate S. aureus-induced skin disease, and provide a potential therapeutic target for treatment of AD.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

BACKGROUND: The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. OBJECTIVE: We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. METHODS: Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. RESULTS: We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. CONCLUSIONS: The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease.

Autonomous SHIP-dependent FcgammaR signaling in pre-B cells leads to inhibition of cell migration and induction of cell death.

Mature B cells express a single immunoglobulin Fc receptor, FcgammaRIIB, that functions to block downstream signaling by co-aggregated antigen receptors. Co-aggregation of receptors is essential because BCR activated kinases must phosphorylate FcgammaRIIB to recruit SHIP and mediate inhibitory signals. Pre-B cells also express FcgammaRIIB, but since they do not yet express antigen receptor, it is unclear when they are activated physiologically. Here, we demonstrate that aggregation of the FcR on pre-B cells leads to potent inhibitory signaling. Aggregation of the FcR alone leads to downstream effects including the induction of cell death and the blockade of SDF-1 induced migration. The biochemical circuitry that mediates this response is unique because although SHIP is required for this signaling and is phosphorylated upon receptor aggregation, this occurs in the absence of FcgammaRIIB phosphorylation. Results indicate that immune complexes may inhibit B cell production in the bone marrow by antigen non-specific mechanisms.