Effects of topical corticosteroid versus tacrolimus on insulin sensitivity and bone homeostasis in adults with atopic dermatitis-A randomized controlled study.

INTRODUCTION: Topical corticosteroids (TCS), used to treat atopic dermatitis (AD), have been associated with type 2 diabetes and osteoporosis in epidemiological studies, possibly explained by systemic absorption. OBJECTIVES: We examined whether intensive daily whole-body TCS treatment over 2 weeks followed by twice weekly application for 4 weeks could elicit insulin resistance and increase bone resorption in adults with AD. METHODS: A randomized parallel-group double-blind double-dummy non-corticosteroid-based active comparator study design was completed in Copenhagen, Denmark. Thirty-six non-obese, non-diabetic adults with moderate-to-severe AD were randomized to whole-body treatment with betamethasone 17-valerate 0.1% plus a vehicle once daily or tacrolimus 0.1% twice daily after washout. Insulin sensitivity assessed by the hyperinsulinemic-euglycemic clamp combined with tracer infusions and biomarkers of bone formation (P1NP) and resorption (CTX) were evaluated at baseline, after 2 weeks of daily treatment and after further 4 weeks of twice-weekly maintenance treatment. RESULTS: AD severity improved with both treatments and systemic inflammation was reduced. After 2 weeks, we observed similar increase in peripheral insulin sensitivity with use of betamethasone (n = 18) and tacrolimus (n = 18). Bone resorption biomarker, CTX, was unchanged, while bone formation marker, P1NP, decreased after betamethasone treatment after both 2 and 6 weeks but remained unchanged in the tacrolimus arm. CONCLUSIONS: Whole-body treatment with TCS leads to systemic exposure but appears not to compromise glucose metabolism during short-term use, which may be a result of reduced systemic inflammatory activity. The negative impact on bone formation could be regarded an adverse effect of TCS.

Using chemiluminescence imaging of cells (CLIC) for relative protein quantification.

Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.

EGFR modulates complement activation in head and neck squamous cell carcinoma.

BACKGROUND: The epidermal growth factor receptor (EGFR) is pivotal for growth of epithelial cells and is overexpressed in several epithelial cancers like head and neck squamous cell carcinoma (HNSCC). EGFR signalling is also involved in diverse innate immune functions in epithelia. We previously found a role for EGFR in modulating the complement system in skin, this prompted an investigation into EGFR role in complement modulation in HNSCC. METHODS: We used patient derived HNSCC cell lines with varying sensitivities to EGFR inhibitors, and generated EGFR inhibition resistant cell lines to study the role of EGFR in modulating complement in HNSCC. RESULTS: We found that HNSCC cell lines activate the complement system when incubated with human serum. This complement activation was increased in cell lines sensitive to EGFR inhibition following the use of the tyrosine kinase inhibitor Iressa. Sensitive cell line made resistant to EGFR-inhibitors displayed complement activation and a decrease in complement regulatory proteins even in the absence of EGFR-inhibitors. Complement activation did not cause lysis of HNSCC cells, and rather led to increased extracellular signal-regulated kinase (ERK) phosphorylation in one cell line. CONCLUSION: These data indicate that EGFR has a complement modulatory role in HNSCC, and that a prolonged EGFR-inhibition treatment in sensitive cancer cells increases complement activation. This has implications in understanding the response to EGFR inhibitors, in which resistance and inflammatory skin lesions are two major causes for treatment cessation.

Development of a Conceptual Model of Chronic Hand Eczema (CHE) Based on Qualitative Interviews with Patients and Expert Dermatologists.

INTRODUCTION: Chronic hand eczema (CHE) is a relapsing inflammatory dermatologic disease. Signs and symptoms can have a significant impact on patients' health-related quality of life (HRQoL). The aim of this study is to characterize the core signs, symptoms and impacts of CHE to develop a conceptual model. METHODS: A structured literature search and qualitative interviews with 20 adult CHE patients in the US and 5 expert dermatologists were conducted to explore the patient experience of CHE signs, symptoms and impacts. Findings were used to support the development of a conceptual model. RESULTS: There was a paucity of CHE qualitative research in the literature, supporting the need for the prospective qualitative research. The primary signs and symptoms identified from the literature review and interviews included itch, dryness, cracking, pain, thickened skin and bleeding. The most salient impacts included embarrassment and appearance concerns, frustration, impacts on work and sleep disturbance. Saturation was achieved for all signs, symptoms and impact concepts. CONCLUSIONS: Findings from this literature review and in-depth qualitative interviews supported the development of a comprehensive conceptual model documenting the signs, symptoms and impacts relevant to CHE patients. Such a model is of considerable value given the lack of existing studies in the literature focused on the qualitative exploration of the CHE patient experience. Limitations included the patient sample being only from the US and not including some CHE subtypes.

Persistent Intracellular Staphylococcus aureus in Keratinocytes Lead to Activation of the Complement System with Subsequent Reduction in the Intracellular Bacterial Load.

The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms-important in causing chronic and therapy resistant infections-is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.

NETQUANT: Automated Quantification of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) that are extensive webs of DNA covered with antimicrobial proteins into the extracellular environment during infection or inflammation as a part of their defense arsenal. Image acquisition of fluorescently labeled NETs and subsequent image-based quantification is frequently used to analyze NET formation (NETosis) in response to various stimuli. However, there are important limitations in the present methods for quantification. Manual methods tend to be error-prone, tedious, and often quite subjective, whereas the software-rooted options are either semi-automatic or difficult to operate. Here, we present an automated and uncomplicated approach for quantifying NETs from fluorescence images, built as a freely available app for MATLAB®. It is based on detection of a set of clearly defined parameters, all related to the biological manifestation of NETs and allowing for single-cell resolution quantification and analysis.

Neutrophil extracellular traps - the dark side of neutrophils.

Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those originally described in vitro. Citrullination of histones by peptidyl arginine deiminase 4 (PAD4) is central for NET formation in vivo. NETs may spur formation of autoantibodies and may also serve as scaffolds for thrombosis, thereby providing a link among infection, autoimmunity, and thrombosis. In this review, we present the mechanisms by which NETs are formed and discuss the physiological and pathophysiological consequences of NET formation. We conclude that NETs may be of more importance in autoimmunity and thrombosis than in innate immune defense.

A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa.

Neutrophils are essential for host defense at the oral mucosa and neutropenia or functional neutrophil defects lead to disordered oral homeostasis. We found that neutrophils from the oral mucosa harvested from morning saliva had released neutrophil extracellular traps (undergone NETosis) in vivo. The NETosis was mediated through intracellular signals elicited by binding of sialyl Lewis(X) present on salival mucins to l-selectin on neutrophils. This led to rapid loss of nuclear membrane and intracellular release of granule proteins with subsequent neutrophil extracellular trap (NET) release independent of elastase and reduced NAD phosphate-oxidase activation. The saliva-induced NETs were more DNase-resistant and had higher capacity to bind and kill bacteria than NETs induced by bacteria or by phorbol-myristate acetate. Furthermore, saliva/sialyl Lewis(X) mediated signaling enhanced intracellular killing of bacteria by neutrophils. Saliva from patients with aphthous ulcers and Behçet disease prone to oral ulcers failed to induce NETosis, but for different reasons it demonstrated that disordered homeostasis in the oral cavity may result in deficient saliva-mediated NETosis.

Platelet-Dependent Neutrophil Function Is Dysregulated by M Protein from Streptococcus pyogenes.

Platelets are rapidly responsive sentinel cells that patrol the bloodstream and contribute to the host response to infection. Platelets have been reported to form heterotypic aggregates with leukocytes and may modulate their function. Here, we have investigated platelet-neutrophil complex formation and neutrophil function in response to distinct agonists. The endogenous platelet activator thrombin gave rise to platelet-dependent neutrophil activation, resulting in enhanced phagocytosis and bacterial killing. Streptococcus pyogenes is an important causative agent of severe infectious disease, which can manifest as sepsis and septic shock. M1 protein from S. pyogenes also mediated platelet-neutrophil complex formation; however, these neutrophils were dysfunctional and exhibited diminished chemotactic ability and bacterial killing. This reveals an important agonist-dependent neutrophil dysfunction during platelet-neutrophil complex formation and highlights the role of platelets during the immune response to streptococcal infection.

A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation.

α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components.

Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses.

Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.

The epidermal growth factor receptor is a regulator of epidermal complement component expression and complement activation.

The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin wounds in vivo and in cultured keratinocytes after exposure to supernatant from stimulated mononuclear cells. In contrast, the epidermal expression of complement components was downregulated in ex vivo injured skin lacking the stimulation from infiltrating inflammatory cells but with intact injury-induced epidermal growth factor receptor (EGFR)-mediated growth factor response. In cultured primary keratinocytes, stimulation with the potent EGFR ligand, TGF-α, yielded a significant downregulation of complement component expression. Indeed, EGFR inhibition significantly enhanced the induction of complement components in keratinocytes and epidermis following stimulation with proinflammatory cytokines. Importantly, EGFR inhibition of cultured keratinocytes either alone or in combination with proinflammatory stimulus promoted activation of the complement system after incubation with serum. In keratinocytes treated solely with the EGFR inhibitor, complement activation was dependent on serum-derived C1q, whereas in keratinocytes stimulated with a combination of proinflammatory cytokines and EGFR inhibition, complement activation was found even with C1q-depleted serum. In contrast to human keratinocytes, EGFR inhibition did not enhance complement component expression or cause complement activation in murine keratinocytes. These data demonstrate an important role for EGFR in regulating the expression of complement components and complement activation in human epidermis and keratinocytes and, to our knowledge, identify for the first time a pathway important for the epidermal regulation of complement activation.

Transcription factor binding site analysis identifies FOXO transcription factors as regulators of the cutaneous wound healing process.

The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.

The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis.

Sepsis is characterized by a dysregulated host-pathogen response, leading to high cytokine levels, excessive coagulation and failure to eradicate invasive bacteria. Novel therapeutic strategies that address crucial pathogenetic steps during infection are urgently needed. Here, we describe novel bioactive roles and therapeutic anti-infective potential of the peptide EDC34, derived from the C-terminus of tissue factor pathway inhibitor-2 (TFPI-2). This peptide exerted direct bactericidal effects and boosted activation of the classical complement pathway including formation of antimicrobial C3a, but inhibited bacteria-induced activation of the contact system. Correspondingly, in mouse models of severe Escherichia coli and Pseudomonas aeruginosa infection, treatment with EDC34 reduced bacterial levels and lung damage. In combination with the antibiotic ceftazidime, the peptide significantly prolonged survival and reduced mortality in mice. The peptide's boosting effect on bacterial clearance paired with its inhibiting effect on excessive coagulation makes it a promising therapeutic candidate for invasive Gram-negative infections.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

ß-Microseminoprotein endows post coital seminal plasma with potent candidacidal activity by a calcium- and pH-dependent mechanism.

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70-75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as ß-microseminoprotein. At neutral pH, the fungicidal activity of ß-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E(71) was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of ß-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine ß-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the ß-microseminoprotein family. By electron microscopy, ß-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that ß-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify ß-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities.

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.

Tissue factor pathway inhibitor 2 is found in skin and its C-terminal region encodes for antibacterial activity.

BACKGROUND: Tissue factor pathway inhibitor 2 (TFPI-2) is a matrix-associated serine protease inhibitor with an enigmatic function in vivo. Here, we describe that TFPI-2 is present in fibrin of wounds and also expressed in skin, where it is up-regulated upon wounding. METHODOLOGY AND PRINCIPAL FINDINGS: Neutrophil elastase cleaved TFPI-2, and a C-terminal fragment was found to bind to bacteria. Similarly, a prototypic peptide representing this C-terminal part, EDC34, bound to bacteria and bacterial lipopolysaccharide, and induced bacterial permeabilization. The peptide also induced leakage in artificial liposomes, and displayed a random coil conformation upon interactions with liposomes as well as lipopolysaccharide. EDC34 was antibacterial against both Gram-negative and Gram-positive bacteria in physiological buffer conditions. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that the C-terminus of TFPI-2 encodes for antimicrobial activity, and may be released during wounding.

Thymic stromal lymphopoietin exerts antimicrobial activities.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.

Up-regulation of A1M/α1-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage.

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (K(d) = 0.96×10(-6) M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.