Jürgen Schauber

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D–dependent mechanism

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

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)

Antimicrobial peptides and the skin immune defense system

Our skin is constantly challenged by microbes but is rarely infected. Cutaneous production of antimicrobial peptides (AMPs) is a primary system for protection, and expression of some AMPs further increases in response to microbial invasion. Cathelicidins are unique AMPs that protect the skin through 2 distinct pathways: (1) direct antimicrobial activity and (2) initiation of a host response resulting in cytokine release, inflammation, angiogenesis, and reepithelialization. Cathelicidin dysfunction emerges as a central factor in the pathogenesis of several cutaneous diseases, including atopic dermatitis, in which cathelicidin is suppressed; rosacea, in which cathelicidin peptides are abnormally processed to forms that induce inflammation; and psoriasis, in which cathelicidin peptide converts self-DNA to a potent stimulus in an autoinflammatory cascade. Recent work identified vitamin D3 as a major factor involved in the regulation of cathelicidin. Therapies targeting control of cathelicidin and other AMPs might provide new approaches in the management of infectious and inflammatory skin diseases.

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.

Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions

In psoriasis, cytosolic DNA in keratinocytes triggers maturation of the cytokine IL-1β via the AIM2 inflammasome. Taking AIM at Psoriasis Everyday life is filled with dangers—from road construction and wet floors to biohazards and radiation. Warning signs, whether the ubiquitous ‘!’ or the classic skull and crossbones, signal impending peril, giving us the chance to change our course of action to avoid the hazards. Cells in the body also respond to danger signals, such as cytosolic DNA, which warns of microbial presence, activating inflammasomes that ramp up the body’s inflammatory response to damage. Now, Dombrowski et al. implicate cytosolic DNA in the inflammation associated with psoriasis, an autoimmune skin disease, and suggest how vitamin D may be able to counteract this response. Psoriasis is a chronic inflammatory disease that commonly causes red, itchy plaques on the skin. Dombrowski et al. inspected both psoriatic and healthy human skin and found higher amounts of cytosolic DNA and AIM2—a DNA sensor associated with inflammasomes—in psoriatic keratinocytes, the predominant cell type in skin. Cytosolic DNA activated AIM2 inflammasomes in keratinocytes, which resulted in the production of proinflammatory cytokines. Importantly, the authors found that the cathelicidin peptide LL-37, which has antimicrobial function, also served as an anti-inflammatory agent by blocking activation of the DNA-sensing inflammasomes. Vitamin D controls cathelicidin production in human skin, and both topical vitamin D treatment and ultraviolet (UV) B light, which up-regulates vitamin D, are currently used to treat psoriasis. The new data from Dombrowski et al. not only provide a mechanistic explanation for the success of these therapies but also suggest that cathelicidin may constitute a new target that specifically affects the inflammasome. For psoriasis, the danger signal itself may be the hazard, and cathelicidin could be a means of defense. The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

IL-17A Enhances Vitamin D3-Induced Expression of Cathelicidin Antimicrobial Peptide in Human Keratinocytes

Cathelicidin is strongly expressed in lesional skin in psoriasis and may play an important role as both an antimicrobial peptide and as an autoinflammatory mediator in this chronic skin disease. The mechanism of increased cathelicidin in psoriatic keratinocytes is not known, but recent observations have found that psoriasis has abundant Th17 cells that produce IL-17A and IL-22. We found that human keratinocytes stimulated with supernatants from T cells isolated from lesional psoriatic skin increased expression of cathelicidin when stimulated in the presence of 1,25-dihydroxyvitamin D3 (1,25D3). This increase was signaled through the IL-17RA. In vitro, IL-17A, but not IL-22, enhanced cathelicidin mRNA and peptide expression in keratinocytes dependent on the presence of 1,25D3. At the same time, coincubation with 1,25D3 blocked induction of human β-defensin 2 (HBD2), IL-6, and IL-8, which are other target genes of IL-17A. Act1, an adaptor associated with IL-17RA and essential for IL-17A signaling, mediated cathelicidin induction, as its suppression by small interfering RNA inhibited HBD2 and cathelicidin. Both, 1,25D3 and IL-17A signaled cathelicidin induction through MEK-ERK. These results suggest that increased IL-17A in psoriatic skin increases cathelicidin through a vitamin D3-, Act1-, and MEK-ERK-dependent mechanism. Therapy targeting this cathelicidin-regulating system might be beneficial in patients suffering from psoriasis.

New insights into rosacea pathophysiology: A review of recent findings

Rosacea is a common, chronic inflammatory skin disease of poorly understood origin. Based on its clinical features (flushing, chronic inflammation, fibrosis) and trigger factors, a complex pathobiology involving different regulatory systems can be anticipated. Although a wealth of research has shed new light over recent years on its pathophysiology, the precise interplay of the various dysregulated systems (immune, vascular, nervous) is still poorly understood. Most authors agree on 4 major clinical subtypes of rosacea: erythematotelangiectatic rosacea, papulopustular rosacea, phymatous rosacea, and ocular rosacea. Still, it needs to be elucidated whether these subtypes develop in a consecutive serial fashion or if any subtypes may occur individually as part of a syndrome. Because rosacea often affects multiple family members, a genetic component is also suspected, but the genetic basis of rosacea remains unclear. During disease manifestation and early stage, the innate immune system and neurovascular dysregulation seem to be driving forces in rosacea pathophysiology. Dissection of major players for disease progression and in advanced stages is severely hampered by the complex activation of the innate and adaptive immune systems, enhanced neuroimmune communication, profound blood vessel and possibly lymphatic vessel changes, and activation of almost every resident cell in the skin. This review discusses some of the recent findings and aims to build unifying hypotheses for a modern understanding of rosacea pathophysiology.

Heterogeneous expression of human cathelicidin hCAP18/LL-37 in inflammatory bowel diseases.

Background Inflammatory bowel diseases (IBDs) are characterized by a breakdown of colon epithelial barrier function. Antimicrobial peptides like cathelicidins are molecules of the innate immune system located at epithelial surfaces. Cathelicidins influence microbial growth and inflammation and may play a role in IBD. In this study, the expression of human cathelicidin hCAP18/LL-37 was investigated in the intestinal mucosa from patients suffering from ulcerative colitis or Crohns disease. Methods Biopsy material from colon and ileal mucosa of a total of 89 patients (34 with Crohns disease, 27 with ulcerative colitis, 28 control patients) was evaluated for cathelicidin expression by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. Colon epithelial cells were stimulated in vitro with various cytokines to evaluate mechanisms that influence cathelicidin production. Results Cathelicidin expression was significantly increased in inflamed and non-inflamed colon mucosa from ulcerative colitis patients compared to non-inflamed control mucosa. In patients with Crohns disease cathelicidin expression was not changed in inflamed or non-inflamed colon or ileal mucosa independent of NOD2 status. Biopsies evaluated by immunohistochemistry showed epithelial cathelicidin expression in the upper crypt that was diffuse in controls and only basal in IBD patients. Inflammation mediators, alone or in combination with the known cathelicidin inducer butyrate, had no effect on cathelicidin expression in cultured colon cells. Conclusions In IBD the colonic expression of human cathelicidin is altered: cathelicidin expression is increased in inflamed and non-inflamed mucosa in patients suffering from ulcerative colitis but not in Crohns disease. This deficiency may further compromise the antimicrobial barrier in Crohns disease.

Narrowband ultraviolet B treatment improves vitamin D balance and alters antimicrobial peptide expression in skin lesions of psoriasis and atopic dermatitis

Background  Narrowband ultraviolet B (NB‐UVB) is a routine treatment for psoriasis and atopic dermatitis (AD) but its effect on vitamin D balance is not well studied.

Defensins and cathelicidins in gastrointestinal infections.

Purpose of review To review recently published studies presenting novel and relevant information on antimicrobial peptides in gastrointestinal infections. Recent findings Defensins and cathelicidins are important antimicrobial peptides expressed by the gastrointestinal epithelium. Their localization and regulation have been the focus of current research establishing the relevance of these peptides both in counteracting an attack by pathogens as well as in controlling the endogenous bacterial flora. In the small intestine, Paneth cell α-defensins maintain a low level of microorganisms and regulate the composition of the bacterial flora. In contrast, a constitutive β-defensin can be found in nearly all gastrointestinal tissues. Other relevant β-defensins as well as human cathelicidin are inducible by inflammation or infections. Thus Helicobacter pylori enhances defensin expression in the gastric mucosa and Campylobacter jejuni and Salmonella provoke a similar response in the colon. Other pathogenic bacteria may suppress the antimicrobial peptide response as an escape strategy. Notably, the therapeutic induction of cathelicidins alleviates experimental shigellosis, suggesting a future role of endogenous antibiotics in medical therapy. Summary These recent findings together with a better understanding of underlying mechanisms involved in the regulation and biology of antimicrobial peptides will open up new therapeutic avenues to battle infections.