Is it possible to diagnose the therapeutic adherence of patients with COPD in clinical practice? A cohort study

Background

Progressive airway inflammation and susceptibility to the airway colonisation and infection are characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD). Antimicrobial peptides (AMPs) are central to the function of the innate host immune response against microbial pathogens and are regulators of inflammation and immunity. S100A7/psoriasin, a recently described AMP, is an essential component of the human epithelia against invading pathogens and acts as an effector molecule of the host innate defence in the skin. We hypothesized that S100A7/psoriasin is involved in the airway mucosal immunity and differently regulated and expressed in the lung during progression of COPD.

Methods

S100A7/psoriasin gene expression was assessed in bronchial biopsies and bronchoalveolar lavage (BAL) fluid cells of healthy controls and COPD patients. Using confocal microscopy and immunohistochemistry, the protein expression of S100A7/psoriasin was investigated.

Results

Here, we report that S100A7/psoriasin, the major antimicrobial peptide of the human skin, is constitutively expressed in perinuclear granules of human bronchial epithelial cells and alveolar macrophages. Whereas typical activators of the innate immune response like TLR ligands and cytokines induced the upregulation of CXCL-8 mRNA and release of CXCL-8 by epithelial cells, S100A7/psoriasin mRNA expression was not modulated. To investigate a potential association of S100A7/psoriasin with COPD, S100A7/psoriasin mRNA expression was assessed in bronchial biopsies and BAL fluid cells of patients at different stages of COPD and controls. Overall, 10 healthy individuals and 34 COPD patients were enrolled in this study. We found an association of S100A7/psoriasin mRNA expression with bacterial detection in the tracheobronchial system (p = 0.0304), which was the strongest in individuals positive for with S. aureus (p = 0.0005). However, S100A7/psoriasin mRNA expression was not altered during the progression of COPD.

Conclusions

S100A7/psoriasin gene expression is unchanged in the airways during COPD. The newly identified association of S100A7/psoriasin with S. aureus may provide new insights into the antimicrobial defence response of the human airways, leading to the induction of S100A7/psoriasin upon microbial challenge.     

Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

Expression of natural antimicrobial peptide beta-defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers

Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of beta-defensin-2 (hBD2), GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC) in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control). Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin-avidin-peroxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p < 0.05). Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05). Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.     

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

Polo-like kinase 1 (Plk1) in non-melanoma skin cancers

Polo-like kinase 1 (Plk1) is becoming an increasingly attractive target for cancer management. Plk1 has been shown to be over-expressed in a variety of cancers; however its role in skin cancers is not well-understood. We recently demonstrated that Plk1 is over-expressed in human melanoma and gene-knockdown as well as chemical-inhibition of Plk1 resulted in a significant decrease in melanoma cell viability and growth without affecting the growth of the normal human epidermal melanocytes (NHEMs). Further, the observed anti-proliferative response of Plk1 was found to be accompanied with a significant G2/M cell cycle arrest, mitotic catastrophe and induction of apoptosis in melanoma cells. In this study, we determined the expression profile of Plk1 in non-melanoma skin cancers viz. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Our data demonstrated that like melanoma, Plk1 is significantly over-expressed in BCC and SCC samples. Further, we also found that compared to normal human epidermal keratinocytes (NHEKs), Plk1 was over-expressed at both the protein and mRNA levels in squamous A253 and A431 cells. In addition, a similar protein expression pattern was found for the downstream targets of Plk1, viz. Cdk1, Cyclin B1 and Cdc25C. We believe that the expression pattern of Plk1 in the various skin cancers, the insusceptibility of normal keratinocytes, to Plk1 inhibition and the easy accessibility for topical applications lends the skin as an attractive tissue for Plk1 based cancer chemoprevention and chemotherapeutic applications.     

Keratinocyte growth factor induces gene expression signature associated with suppression of malignant phenotype of cutaneous squamous carcinoma cells

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.     

Evaluation of aneuploidy frequency for chromosomes 6 and 17 in eyelid tumours using the FISH technique

Although basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are very common skin tumours, the incidence of chromosome aneuploidy with regard to the eyelid has not been investigated. We aimed to find the frequency of chromosome 6 and 17 aneuploidies in eyelid tumours' interphase nuclei with fluorescence in situ hybridization (I-FISH) with chromosome specific DNA probes. I-FISH with chromosome 6 and 17 centromere specific DNA probes was used in the eyelids of 10 patients with BCC or SCC and the peripheral blood cells of 10 healthy donors as controls. The frequency of chromosome 6 and 17 aneuploidies was significantly higher in 7 out of 10 patients and 5 out of 10 patients, respectively, than in controls, indicating a higher frequency of aneuploidy in BCC than in SCC of the eyelid. Distribution of hybridization signals for chromosome 6 and 17 was wide ranging, indicating heterogeneity of cell populations with aneuploidy between patients. These findings indicate that acquisition of chromosome aneuploidies in eyelid tumours may have an important pathogenic role in both BCC and SCC of the eyelid area.     

Human β-D-3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid

Human β-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six β-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-β in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-β. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-β, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.     

Differences in mortality, growth, lysozyme, and toll-like receptor gene expression among genetic groups of catfish exposed to virulent Edwardsiella ictaluri

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.     

CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium

The induction of host antimicrobial molecules following binding of pathogen components to pattern recognition receptors such as CD14 and the Toll-like receptors (TLRs) is a key feature of innate immunity. The human airway epithelium is an important environmental interface, but LPS recognition pathways have not been determined. We hypothesized that LPS would trigger beta-defensin (hBD2) mRNA in human tracheobronchial epithelial (hTBE) cells through a CD14-dependent mechanism, ultimately activating NF-kappa B. An average 3-fold increase in hBD2 mRNA occurs 24 h after LPS challenge of hTBE cells. For the first time, we demonstrate the presence of CD14 mRNA and cell surface protein in hTBE cells and show that CD14 neutralization abolishes LPS induction of hBD2 mRNA. Furthermore, we demonstrate TLR mRNA in hTBE cells and NF-kappa B activation following LPS. Thus, LPS induction of hBD2 in hTBE cells requires CD14, which may complex with a TLR to ultimately activate NF-kappa B.     

Expression analysis of turkey (Meleagris gallopavo) toll-like receptors and molecular characterization of avian specific TLR15

Toll-like receptors (TLRs) constitute a multi-gene family, which plays a pivotal role in sensing invading pathogens by virtue of conserved microbial patterns. TLR repertoire of chicken and zebra finch has been well studied. However TLR family of other avian species is yet to be characterized. In the present study, we identified TLR repertoire of turkey, characterized avian specific receptor TLR15 in turkey and profiled the TLRs expressions in a range of tissues of turkey poults. All ten TLR genes orthologous to chicken TLR repertoire were found in turkey. Turkey TLR genes showed 81-93 % similarity at amino acid level to their chicken counter parts. Phylogenetic analysis confirmed the orthologous relationship of turkey TLRs with chicken and zebra finch TLRs. Open reading frame of turkey TLR15 was 2,607 bp long encoding 868 amino acids similar to that of broiler chicken and showed 92.4, 91.1 and 69.5 % identity at amino acid levels with chicken, Japanese quail and zebra finch TLR15 sequences respectively. Overall TLR expression was highest for TLR4 and lowest for TLR21. TLR1A, 2A, 2B and 21 were significantly higher in liver than other tissues investigated (P < 0.01). TLR3 expression was significantly higher in bone marrow (BM) and spleen in comparison to other tissues studied (P < 0.01). Furthermore, no significant differences in the expression levels of TLR1B, 4, 5, 7 and 15 genes were detected among the tissues studied. Our findings contribute to the characterization of innate immune system of birds and show the innate preparedness of young turkey poults to a range of pathogens.     

Hip to the Game: YAP/TAZ is required for nonmelanoma skin cancers

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the two most common skin cancers found in humans. These cancers can acquire drug resistance and pose considerable medical burdens to clinics and patients if left untreated. Two recent studies show that active Hippo signaling plays a critical role in initiating BCC and SCC tumorigenesis, providing new opportunities to develop therapies against these skin malignancies.     

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.