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1.  Etanercept decreases the innate immune wounding response in psoriasis 
Experimental dermatology  2013;22(9):599-601.
Cathelicidin is increased when normal skin is injured and in psoriasis lesions where it has been suggested to play a pivotal role in inflammation through interactions with self-DNA and toll-like receptor 9 (TLR-9) in keratinocytes and plasmacytoid dendritic cells. Because of etanercept’s success in treating psoriasis, we hypothesized that etanercept may suppress TLR-9 and cathelicidin induction. Examination of experimentally induced wounds of psoriatic lesional and non-lesional skin, and comparison with wounded normal skin, shows that the induction of cathelicidin and TLR-9 is greatly enhanced in lesional psoriatic skin. Six weeks of etanercept appears not to affect the baseline expression of cathelicidin or TLR-9, but does blunt the induction of cathelicidin in psoriasis with wounding. These findings support the role of cathelicidin in the enhancement of local inflammation in psoriasis and may partially explain one of the mechanisms enabling TNF-α inhibitors to successfully treat this disorder.
doi:10.1111/exd.12167
PMCID: PMC4195436  PMID: 23802569
cathelicidin; etanercept; psoriasis; TLR-9
2.  Dermal adipocytes protect against invasive Staphylococcus aureus skin infection 
Science (New York, N.Y.)  2015;347(6217):67-71.
Adipocytes have been suggested to be immunologically active, but their role in host defense is unclear. We observed rapid proliferation of preadipocytes and expansion of the dermal fat layer after infection of the skin by Staphylococcus aureus. Impaired adipogenesis resulted in increased infection as seen in Zfp423nur12 mice or in mice given inhibitors of peroxisome proliferator–activated receptor γ. This host defense function was mediated through the production of cathelicidin antimicrobial peptide from adipocytes because cathelicidin expression was decreased by inhibition of adipogenesis, and adipocytes from Camp−/− mice lost the capacity to inhibit bacterial growth. Together, these findings show that the production of an antimicrobial peptide by adipocytes is an important element for protection against S. aureus infection of the skin.
doi:10.1126/science.1260972
PMCID: PMC4318537  PMID: 25554785
3.  Doxycycline Indirectly Inhibits Proteolytic Activation of Tryptic Kallikrein-Related Peptidases and Activation of Cathelicidin 
The increased abundance and activity of cathelicidin and kallikrein 5 (KLK5), a predominant trypsin-like serine protease (TLSP) in the stratum corneum, have been implicated in the pathogenesis of rosacea, a disorder treated by the use of low-dose doxycycline. Here we hypothesized that doxycycline can inhibit activation of tryptic KLKs through an indirect mechanism by inhibition of matrix metalloproteinases (MMPs) in keratinocytes. The capacity of doxycycline to directly inhibit enzyme activity was measured in surface collections of human facial skin and extracts of cultured keratinocytes by fluorescence polarization assay against fluorogenic substrates specific for MMPs or TLSPs. Doxycycline did inhibit MMP activity but did not directly inhibit serine protease activity against a fluorogenic substrate specific for TLSPs. However, when doxycycline or other MMP inhibitors were added to live keratinocytes during the production of tryptic KLKs, this treatment indirectly resulted in decreased TLSP activity. Furthermore, doxycycline under these conditions inhibited the generation of the cathelicidin peptide LL-37 from its precursor protein hCAP18, a process dependent on KLK activity. These results demonstrate that doxycycline can prevent cathelicidin activation, and suggest a previously unknown mechanism of action for doxycycline through inhibiting generation of active cathelicidin peptides.
doi:10.1038/jid.2012.14
PMCID: PMC4169281  PMID: 22336948
4.  Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: Implications of probiotics in acne vulgaris 
Increasing evidence demonstrates that commensal microorganisms in the human skin microbiome help fight pathogens and maintain homeostasis of the microbiome. However, it is unclear how these microorganisms maintain biological balance when one of them overgrows. The overgrowth of Propionibacterium acnes (P. acnes), a commensal skin bacterium, has been associated with the progression of acne vulgaris. Our results demonstrate that skin microorganisms can mediate fermentation of glycerol, which is naturally produced in skin, to enhance their inhibitory effects on P. acnes growth. The skin microorganisms, most of which have been identified as Staphylococcus epidermidis (S. epidermidis), in the microbiome of human fingerprints can ferment glycerol and create inhibition zones to repel a colony of overgrown P. acnes. Succinic acid, one of four short-chain fatty acids (SCFAs) detected in fermented media by nuclear magnetic resonance (NMR) analysis, effectively inhibits the growth of P. acnes in vitro and in vivo. Both intralesional injection and topical application of succinic acid to P. acnes-induced lesions markedly suppress the P. acnes-induced inflammation in mice. We demonstrate for the first time that bacterial members in the skin microbiome can undergo fermentation to rein in the overgrowth of P. acnes. The concept of bacterial interference between P. acnes and S. epidermidis via fermentation can be applied to develop probiotics against acne vulgaris and other skin diseases. In addition, it will open up an entirely new area of study for the biological function of the skin microbiome in promoting human health.
doi:10.1007/s00253-013-5394-8
PMCID: PMC3888247  PMID: 24265031
Acne; Fermentation; P. acnes; Probiotic; S. epidermidis; Skin Microbiome
5.  Innate Immune Sensors Stimulate Inflammatory and Immunosuppressive Responses to UVB Radiation 
Almost 40 years from when it was first reported that UVB radiation exposure would modulate immune signaling, the photoimmunology field is still trying to understand the mechanisms by which UVB initiates inflammatory responses and modulates immune recognition. This commentary focuses on the ability of Toll-like receptors (TLRs), specifically TLR4 (Ahmad et al., 2014) and ligands such as damage-associated molecular patterns (DAMPs) released from injured cells to stimulate innate immune signaling and inflammatory cytokine production following UVB irradiation.
doi:10.1038/jid.2014.32
PMCID: PMC4271625  PMID: 24825061
6.  Cathelicidin-Deficient (Cnlp−/−) Mice Show Increased Susceptibility to Pseudomonas aeruginosa Keratitis 
Purpose
To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice.
Methods
PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 129/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and 21. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1β, IL-6, macrophage inflammatory peptide (MIP)-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) levels in the corneas.
Results
WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by 21 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and 21 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1β, IL-6, MIP-2, KC, TNF-α, and VEGF up to day 21 PI compared with the WT mice.
Conclusions
Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.
doi:10.1167/iovs.07-0274
PMCID: PMC4234056  PMID: 17898271
7.  Functions of the skin microbiota in health and disease 
Seminars in immunology  2013;25(5):370-377.
The skin, the human body’s largest organ, is home to a diverse and complex variety of innate and adaptive immune functions. Despite this potent immune system present at the cutaneous barrier, the skin encourages colonization by microorganisms. Characterization these microbial communities has enhanced our knowledge of the ecology of organisms present in normal skin; furthermore, studies have begun to bring to light the intimate relationships shared between host and resident microbes. In particular, it is apparent that just as host immunological factors and behaviors shape the composition of these communities, microbes present on the skin greatly impact the functions of human immunity. Thus, today the skin immune system should be considered a collective mixture of elements from the host and microbes acting in a mutualistic relationship. In this article we will review recent findings of the interactions of skin microbial communities with host immunity, and discuss the role that dysbiosis of these communities plays in diseases of the skin.
doi:10.1016/j.smim.2013.09.005
PMCID: PMC4219649  PMID: 24268438
Keratinocyte; Staphylococcus; Propionibacterium acnes; Antimicrobial peptides; Dysbiosis
9.  Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel 
Background
Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea.
Objective
We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system.
Methods
Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel.
Results
AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment.
Limitations
Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA.
Conclusions
These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity.
doi:10.1016/j.jaad.2013.05.019
PMCID: PMC3910251  PMID: 23871720
antimicrobial peptides; azelaic acid; cathelicidin; kallikrein 5; LL-37; rosacea; serine protease
10.  Hyaluronan digestion controls DC migration from the skin 
The Journal of Clinical Investigation  2014;124(3):1309-1319.
The breakdown and release of hyaluronan (HA) from the extracellular matrix has been hypothesized to act as an endogenous signal of injury. To test this hypothesis, we generated mice that conditionally overexpressed human hyaluronidase 1 (HYAL1). Mice expressing HYAL1 in skin either during early development or by inducible transient expression exhibited extensive HA degradation, yet displayed no evidence of spontaneous inflammation. Further, HYAL1 expression activated migration and promoted loss of DCs from the skin. We subsequently determined that induction of HYAL1 expression prior to topical antigen application resulted in a lack of an antigenic response due to the depletion of DCs from the skin. In contrast, induction of HYAL1 expression concurrent with antigen exposure accelerated allergic sensitization. Administration of HA tetrasaccharides, before or simultaneously with antigen application, recapitulated phenotypes observed in HYAL1-expressing animals, suggesting that the generation of small HA fragments, rather than the loss of large HA molecules, promotes DC migration and subsequent modification of allergic responses. Furthermore, mice lacking TLR4 did not exhibit HA-associated phenotypes, indicating that TLR4 mediates these responses. This study provides direct evidence that HA breakdown controls the capacity of the skin to present antigen. These events may influence DC function in injury or disease and have potential to be exploited therapeutically for modification of allergic responses.
doi:10.1172/JCI67947
PMCID: PMC3934161  PMID: 24487587
11.  Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation and epidermal organelles 
Injury to the skin, and the subsequent release of non-coding double-stranded RNA from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). Since changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. Double-stranded RNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with double-stranded RNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by oil-red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that double-stranded RNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance.
doi:10.1038/jid.2013.39
PMCID: PMC3686920  PMID: 23353987
12.  HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection 
Nature communications  2013;4:1963.
HSV-1 is an important epithelial pathogen and has the potential for significant morbidity in humans. Here we demonstrate that a cell surface scavenger receptor, macrophage receptor with collagenous structure (MARCO), previously thought to enhance antiviral defense by enabling nucleic acid recognition, is usurped by HSV-1 and functions together with heparan sulfate proteoglycans to mediate adsorption to epithelial cells. Ligands of MARCO dramatically inhibit HSV-1 adsorption and infection of human keratinocytes and protect mice against infection. HSV-1 glycoprotein C (gC) closely co-localizes with MARCO at the cell surface, and gC binds directly to purified MARCO with high affinity. Increasing MARCO expression enhances HSV-1 infection while MARCO-/- mice have reduced susceptibility to infection by HSV-1. These findings demonstrate that HSV-1 binds to MARCO to enhance its capacity for disease, and suggests a new therapeutic target to alter pathogenicity of HSV-1 in skin infection.
doi:10.1038/ncomms2963
PMCID: PMC3681428  PMID: 23739639
13.  Cathelicidin Host Defence Peptide Augments Clearance of Pulmonary Pseudomonas aeruginosa Infection by Its Influence on Neutrophil Function In Vivo 
PLoS ONE  2014;9(6):e99029.
Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.
doi:10.1371/journal.pone.0099029
PMCID: PMC4041793  PMID: 24887410
14.  Neutrophil-derived cathelicidin promotes adhesion of classical monocytes 
Circulation research  2013;112(5):792-801.
Rationale
The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood.
Objective
To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation.
Methods and Results
Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of non-classical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates FPR2 on classical monocytes, resulting in monocytic β1- and β2-integrin conformational change towards an extended, active conformation that allows for adhesion to their respective ligands VCAM-1 and ICAM-1.
Conclusions
These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.
doi:10.1161/CIRCRESAHA.112.300666
PMCID: PMC3702173  PMID: 23283724
Neutrophil; monocyte; inflammation; cathelicidin; recruitment
15.  Host Immune Defense Peptide LL-37 Activates Caspase-Independent Apoptosis and Suppresses Colon Cancer 
Cancer research  2012;72(24):6512-6523.
Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin–sensitive G-protein–coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG–mediated apoptosis in colon cancer cells.
doi:10.1158/0008-5472.CAN-12-2359
PMCID: PMC3910284  PMID: 23100468
16.  Resveratrol Stimulates Sphingosine-1-Phosphate Signaling of Cathelicidin Production 
We recently discovered a regulatory mechanism that stimulates production of the multifunctional antimicrobial peptide, cathelicidin antimicrobial peptide (CAMP). In response to subtoxic levels of ER stress, increased sphingosine-1-phosphate (S1P) production activates an NFκB→C/EBPα dependent pathway that enhances CAMP production in cultured human keratinocytes. Since the multifunctional stilbenoid compound, resveratrol (RESV), increases ceramide (Cer) levels, a precursor of S1P, we hypothesized and assessed whether RESV could exploit the same pathway to regulate CAMP production. Accordingly, RESV significantly increased Cer and S1P levels in cultured keratinocytes, paralleled by increased CAMP mRNA/protein expression. Furthermore, topical RESV also increased murine CAMP mRNA/protein expression in mouse skin. Conversely, blockade of Cer→sphingosine→S1P metabolic conversion, with specific inhibitors of ceramidase or sphingosine kinase, attenuated the expected RESV-mediated increase in CAMP expression. The RESV-induced increase in CAMP expression required both NF-κB and C/EBPα transactivation. Moreover, conditioned media from keratinocyte treated with RESV significantly suppressed Staphylococcus aureus growth. Finally, topical RESV, if not coapplied with a specific inhibitor of sphingosine kinase, blocked Staphylococcus aureus invasion into murine skin. These results demonstrate that the dietary stilbenoid, RESV, stimulates S1P signaling of CAMP production through an NF-κB→C/EBPα-dependent mechanism, leading to enhanced antimicrobial defense against exogenous microbial pathogens.
doi:10.1038/jid.2013.133
PMCID: PMC3753186  PMID: 23856934
antimicrobial peptide; cathelicidin antimicrobial protein; keratinocytes; resveratrol; sphingosine-1-phosphate
17.  Antimicrobial peptides: Old Molecules with New Ideas 
Almost 90 years have passed since Alexander Fleming discovered the antimicrobial activity of lysozyme, the first natural antibiotic isolated from our body. Since then, various types of molecules with antibiotic activity have been isolated from animals, insects, plants and bacteria, and their use has revolutionised clinical medicine. So far, more than 1200 types of peptides with antimicrobial activity have been isolated from various cells and tissues, and it appears all living organisms employ these antimicrobial peptides (AMPs) in their host defense. In the last decade, innate AMPs produced by mammals have been shown to be essential for the protection of skin and other organs. Their importance is due to their pleiotrophic functions to not only kill microbes but also control host physiological functions such as inflammation, angiogenesis and wound healing. Recent advances in our understanding of the function of AMPs have associated their altered production with various human diseases such as psoriasis, atopic dermatitis and rosacea. In this review, we summarize the history of AMP biology and provide an overview of recent research progress in this field.
doi:10.1038/jid.2011.387
PMCID: PMC3279605  PMID: 22158560
18.  Microbial Symbiosis with the Innate Immune Defense System of the Skin 
Skin protects itself against infection through a variety of mechanisms. Antimicrobial peptides (AMPs) are major contributors to cutaneous innate immunity, and this system, combined with the unique ionic, lipid and physical barrier of the epidermis is the first line defense against invading pathogens. However, recent studies have revealed that our skin’s innate immune system is not solely of human origin. Staphylococcus epidermidis, a major constituent of the normal microflora on healthy human skin, acts as a barrier against colonization of potentially pathogenic microbes and against overgrowth of already present opportunistic pathogens. Our resident commensal microbes produce their own AMPs, act to enhance the normal production of AMPs by keratinocytes, and are beneficial to maintaining inflammatory homeostasis by suppressing excess cytokine release after minor epidermal injury. These observations indicate that the normal human skin microflora protects skin via various modes of action, a conclusion supported by many lines of evidence associating diseases such as acne, atopic dermatitis, psoriasis and rosacea with an imbalance of the microflora even in the absence of classical infection. This review highlights recent observations on the importance of innate immune systems and the relationship with the normal skin microflora to maintain healthy skin.
doi:10.1038/jid.2011.182
PMCID: PMC3174284  PMID: 21697881
19.  The Antimicrobial Protein REG3A Regulates Keratinocyte Proliferation and Differentiation after Skin Injury 
Immunity  2012;37(1):10.1016/j.immuni.2012.04.010.
Summary
Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by kerati-nocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.
doi:10.1016/j.immuni.2012.04.010
PMCID: PMC3828049  PMID: 22727489
20.  Endogenous cathelicidin production limits inflammation and protective immunity to Mycobacterium avium in mice 
The production of antimicrobial peptides, such as the cathelicidins, plays a prominent role in the innate immune response against microbial pathogens. Cathelicidins are widely distributed amongst living organisms, and the antimicrobial peptides generated by proteolysis of the precursor forms are typically cationic and α-helical, a structure that facilitates their interaction and insertion into anionic bacterial cell walls and membranes, causing damage and promoting microbial death. Here, we found that mouse cathelicidin (Camp) expression was induced in bone marrow-derived macrophages by infection with Mycobacterium avium in a TLR2- and TNF-dependent manner. However, the endogenous production of the cathelin-related antimicrobial peptide (CRAMP) was not required for the bacteriostasis of M. avium either in primary cultures of macrophages or in vivo, as shown by the use of CRAMP-null mice. In contrast, the lack of Camp led to a transient improvement of M. avium growth control in the spleens of infected mice while at the same time causing an exacerbation of the inflammatory response to infection. Our data highlight the anti-inflammatory effects of CRAMP and suggests that virulent mycobacteria may possess strategies to escape its antimicrobial activity.
doi:10.1002/iid3.7
PMCID: PMC4220664  PMID: 25400920
Antimicrobial peptides; cathelicidin; macrophage
21.  Ultraviolet Radiation Damages Self Noncoding RNA And Is Detected By TLR3 
Nature medicine  2012;18(8):10.1038/nm.2861.
Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3−/− mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation–induced immune suppression. These findings establish that UVB damage is detected by TLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.
doi:10.1038/nm.2861
PMCID: PMC3812946  PMID: 22772463
22.  Cyclooxygenase-2 Enhances Antimicrobial Peptide Expression and Killing of Staphylococcus aureus 
Antimicrobial peptides such as human β-defensins (hBDs) and cathelicidins are critical for protection against infection and can be induced by activation of TLRs, a pathway that also activates cyclooxygenase(Cox)-2 expression. We hypothesized that Cox-2 is induced by TLR activation and is necessary for optimal AMP production, and that inhibitors of Cox-2 may therefore inhibit antimicrobial action. Normal human keratinocytes (NHEKs) stimulated with a TLR2/6 ligand, macrophage-activating lipo-peptide-2, or a TLR3 ligand, polyinosinic-polycytidylic acid, increased Cox-2 mRNA and protein and increased PGE2, a product of Cox-2. Treatment with a Cox-2 selective inhibitor (SC-58125) or Cox-2 small interfering RNA attenuated hBD2 and hBD3 production in NHEKs when stimulated with macrophage-activating lipopeptide-2, polyinosinic-polycytidylic acid, or UVB (15 mJ/cm2), but it did not attenuate vitamin D3-induced cathelicidin. SC-58125 also inhibited TLR-dependent NF-κB activation. Conversely, treatment with Cox-derived prostanoids PGD2 or 15-deoxy-Δ12,14-PGJ2 induced hBD3 or hBD2 and hBD3, respectively. The functional significance of these observations was seen in NHEKs that showed reduced anti-staphylococcal activity when treated with a Cox-2 inhibitor. These findings demonstrate a critical role for Cox-2 in hBD production and suggest that the use of Cox-2 inhibitors may adversely influence the risk for bacterial infection.
doi:10.4049/jimmunol.1002009
PMCID: PMC3025174  PMID: 20971925
23.  Activation of epidermal toll-like receptor 2 enhances tight junction function – Implications for atopic dermatitis and skin barrier repair 
Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus (S. aureus) skin infections. S. aureus is sensed by many pattern recognition receptors including toll-like receptor (TLR) 2. We hypothesized that an effective innate immune response will include skin barrier repair and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, CLDN1, CLDN23, occludin and ZO-1 in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape-stripping. Tlr2−/− mice had a delayed and incomplete barrier recovery following tape-stripping. AD subjects had reduced epidermal TLR2 expression as compared to nonatopic (NA) subjects, which inversely correlated (r= 0.654, P= 0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may play a role in their incompetent skin barrier.
doi:10.1038/jid.2012.437
PMCID: PMC3600383  PMID: 23223142
24.  The antimicrobial peptide cathelicidin modulates Clostridium difficile-associated colitis and toxin A-mediated enteritis in mice 
Gut  2012;62(9):1295-1305.
Background
Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties.
Objectives
To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes.
Design
Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP.
Results
Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice.
Conclusion
Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.
doi:10.1136/gutjnl-2012-302180
PMCID: PMC3737259  PMID: 22760006
25.  The coordinated Response of the Physical and Antimicrobial Peptide Barriers of the Skin 
Antimicrobial peptides (AMPs) are an essential and multifunctional element for immune defense of the skin during infection and injury. In this issue, Ahrens et al. characterize the response of β-defensins, a class of AMPs, following acute and chronic challenges to the permeability barrier of the skin. Their findings suggest that the antimicrobial and permeability barriers of the skin are closely linked.
doi:10.1038/jid.2010.360
PMCID: PMC3121688  PMID: 21228809

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