Kenshi Yamasaki

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.

Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin

The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL‐37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL‐37 were identified in normal skin. Through the use of selective inhibitors, SELDI‐TOF‐MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5‐deficent mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.—Yamasaki, K., Schauber, J., Coda, A., Lin, H., Dorschner, R. A., Schechter, N. M., Bonnart, C., Descargues, P., Hovnanian, A., Gallo, R. L. Kallikrein‐mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin. FASEB J. 20, 2068–2080 (2006)

Recognition of Hyaluronan Released in Sterile Injury Involves a Unique Receptor Complex Dependent on Toll-like Receptor 4, CD44, and MD-2

Inflammation under sterile conditions is not well understood despite its importance in trauma and autoimmune disease. To investigate this process we established mouse models of sterile injury and explored the role of hyaluronan in mediating inflammation following injury. The response of cultured monocytes to hyaluronan was different than the response to lipopolysaccharide (LPS) despite both being dependent on Toll-like receptor 4 (TLR4). Cultured cells exposed to hyaluronan showed a pattern of gene induction that mimics the response seen in mouse skin after sterile injury with an increase in molecules such as transforming growth factor-β2 and matrix metalloproteinase-13. These factors were not induced by LPS despite the mutual dependence of both hyaluronan and LPS on TLR4. Explanation for the unique response to hyaluronan was provided by observations that a lack of TLR4 or CD44 in mice diminished the response to sterile injury, and together with MD-2, was required for responsiveness to hyaluronan in vitro. Thus, a unique complex of TLR4, MD-2, and CD44 recognizes hyaluronan. Immunoprecipitation experiments confirmed the physical association of TLR4 and CD44. Taken together, our results define a previously unknown mechanism for initiation of sterile inflammation that involves recognition of released hyaluronan fragments as an endogenous signal of tissue injury.

Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli

Immune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and β‐defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D3 induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced β‐defensin expression. In all cell types, vitamin D3 activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D3 activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of β‐defensin‐2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue‐specific manner at transcriptional, post‐transcriptional and post‐translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro‐inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation.

NLRP3/Cryopyrin Is Necessary for Interleukin-1β (IL-1β) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation.

Selective Antimicrobial Action Is Provided by Phenol-Soluble Modulins Derived from Staphylococcus epidermidis, a Normal Resident of the Skin

Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

Nuclear translocation of phosphorylated STAT3 is essential for vascular endothelial growth factor-induced human dermal microvascular endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is a potent, multifunctional, endothelial-cell-specific growth factor. It stimulates proliferation and migration of endothelial cells. Characterization of intracellular signal transduction after VEGF and VEGF receptor (VEGFR) interaction has demonstrated the involvement of the mitogen-activated protein kinase pathway. However, several studies indicated that signal transducers and activators of transcription (STAT) is another important pathway downstream of VEGF/VEGFR interaction. Therefore, we studied the role of STAT3 in the migration and tube formation of the human dermal microvascular endothelial cells (HDMEC). HDMEC expressed phosphorylated forms of STAT1, STAT3, and STAT5, and a marked increase of phosphorylated STAT3 in the nuclear fraction after addition of VEGF was observed by Western blot and immunohistochemical staining. To verify the functional implication of STAT3 phosphorylation in HDMEC migration, we introduced a dominant-negative STAT3 using adenovirus vector system. Dominant-negative STAT3 abolished the VEGF-induced nuclear translocation of phosphorylated STAT3 and inhibited HDMEC migration completely. Dominant-negative STAT3 also suppressed VEGF-induced HDMEC tube formation on Matrigel and on collagen gel. These data demonstrate that STAT3 and its phosphorylation are involved in the downstream pathway of VEGF/VEGFR interaction and regulate VEGF-induced HDMEC migration and tube formation.

TLR2 Expression Is Increased in Rosacea and Stimulates Enhanced Serine Protease Production by Keratinocytes

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea.

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.

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.