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.

The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA), and acts selectively on keratinocytes triggered through Toll-like receptor (TLR) 3. The significance of this is seen by observations that TLR3 activation is required for normal inflammation after injury, and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. These findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.

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-␤2 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.

Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of < 10 kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human β-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.

Cutaneous injury in mice drives transient TLR7- and TLR9-mediated production of type I interferon by plasmacytoid dendritic cells, which is required for re-epithelialization of the skin.

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp−). MCs derived from Cnlp−/− animals had a 50% reduction in their ability to kill group A Streptococcus. These MCs expressed equivalent amounts of mRNA for murine β-defensin-4, a β-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.

The epidermis closely interacts with nerve endings, and both epidermis and nerves produce substances for mutual sustenance. Neuropeptides, like substance P (SP) and calcitonin gene-related protein (CGRP), are produced by sensory nerves in the dermis; they induce mast cells to release vasoactive amines that facilitate infiltration of neutrophils and T cells. Some receptors are more important than others in the generation of itch. The Mas-related G protein-coupled receptors (Mrgpr) family as well as transient receptor potential ankyrin 1 (TRPA1) and protease activated receptor 2(Par2) have important roles in itch and inflammation. The activation of MrgprX1 degranulates mast cells to communicate with sensory nerve and cutaneous cells for developing neurogenic inflammation. Mrgprs and transient receptor potential vanilloid 4 (TRPV4) are crucial for the generation of skin diseases like rosacea, while SP, CGRP, somatostatin, β-endorphin, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) can modulate the immune system during psoriasis development. The increased level of SP, in atopic dermatitis, induces the release of interferon (IFN)-γ, interleukin (IL)-4, tumor necrosis factor (TNF)-α, and IL-10 from the peripheral blood mononuclear leukocytes. We are finally starting to understand the intricate connections between the skin neurons and resident skin cells and how their interaction can be key to controlling inflammation and from there the pathogenesis of diseases like atopic dermatitis, psoriasis, and rosacea.