Human Host Defense Peptide LL-37 Suppresses TNFα-Mediated Matrix Metalloproteinases MMP9 and MMP13 in Human Bronchial Epithelial Cells.

INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.

Airway epithelial cells mount an early response to mycobacterial infection.

Lung epithelial cells represent the first line of host defence against foreign inhaled components, including respiratory pathogens. Their responses to these exposures may direct subsequent immune activation to these pathogens. The epithelial response to mycobacterial infections is not well characterized and may provide clues to why some mycobacterial infections are cleared, while others are persistent and pathogenic. We have utilized an air-liquid interface model of human primary bronchial epithelial cells (ALI-PBEC) to investigate the epithelial response to infection with a variety of mycobacteria: Mycobacterium tuberculosis (Mtb), M. bovis (BCG), M. avium, and M. smegmatis. Airway epithelial cells were found to be infected by all four species, albeit at low frequencies. The proportion of infected epithelial cells was lowest for Mtb and highest for M. avium. Differential gene expression analysis revealed a common epithelial host response to mycobacteria, including upregulation of BIRC3, S100A8 and DEFB4, and downregulation of BPIFB1 at 48 h post infection. Apical secretions contained predominantly pro-inflammatory cytokines, while basal secretions contained tissue growth factors and chemokines. Finally, we show that neutrophils were attracted to both apical and basal secretions of infected ALI-PBEC. Neutrophils were attracted in high numbers to apical secretions from PBEC infected with all mycobacteria, with the exception of secretions from M. avium-infected ALI-PBEC. Taken together, our results show that airway epithelial cells are differentially infected by mycobacteria, and react rapidly by upregulation of antimicrobials, and increased secretion of inflammatory cytokines and chemokines which directly attract neutrophils. Thus, the airway epithelium may be an important immunological component in controlling and regulating mycobacterial infections.

IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways.

BACKGROUND: Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. METHODS: Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and L-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, L-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. RESULTS: CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and L-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. CONCLUSION: Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines. Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

ERS International Congress 2022: highlights from the Basic and Translational Science Assembly.

In this review, the Basic and Translational Science Assembly of the European Respiratory Society provides an overview of the 2022 International Congress highlights. We discuss the consequences of respiratory events from birth until old age regarding climate change related alterations in air quality due to pollution caused by increased ozone, pollen, wildfires and fuel combustion as well as the increasing presence of microplastic and microfibres. Early life events such as the effect of hyperoxia in the context of bronchopulmonary dysplasia and crucial effects of the intrauterine environment in the context of pre-eclampsia were discussed. The Human Lung Cell Atlas (HLCA) was put forward as a new point of reference for healthy human lungs. The combination of single-cell RNA sequencing and spatial data in the HLCA has enabled the discovery of new cell types/states and niches, and served as a platform that facilitates further investigation of mechanistic perturbations. The role of cell death modalities in regulating the onset and progression of chronic lung diseases and its potential as a therapeutic target was also discussed. Translational studies identified novel therapeutic targets and immunoregulatory mechanisms in asthma. Lastly, it was highlighted that the choice of regenerative therapy depends on disease severity, ranging from transplantation to cell therapies and regenerative pharmacology.

Impact of Changes in Human Airway Epithelial Cellular Composition and Differentiation on SARS-CoV-2 Infection Biology.

The consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can range from asymptomatic to fatal disease. Variations in epithelial susceptibility to SARS-CoV-2 infection depend on the anatomical location from the proximal to distal respiratory tract. However, the cellular biology underlying these variations is not completely understood. Thus, air-liquid interface cultures of well-differentiated primary human tracheal and bronchial epithelial cells were employed to study the impact of epithelial cellular composition and differentiation on SARS-CoV-2 infection by transcriptional (RNA sequencing) and immunofluorescent analyses. Changes of cellular composition were investigated by varying time of differentiation or by using specific compounds. We found that SARS-CoV-2 primarily infected not only ciliated cells but also goblet cells and transient secretory cells. Viral replication was impacted by differences in cellular composition, which depended on culturing time and anatomical origin. A higher percentage of ciliated cells correlated with a higher viral load. However, DAPT treatment, which increased the number of ciliated cells and reduced goblet cells, decreased viral load, indicating the contribution of goblet cells to infection. Cell entry factors, especially cathepsin L and transmembrane protease serine 2, were also affected by differentiation time. In conclusion, our study demonstrates that viral replication is affected by changes in cellular composition, especially in cells related to the mucociliary system. This could explain in part the variable susceptibility to SARS-CoV-2 infection between individuals and between anatomical locations in the respiratory tract.

Early transcriptional responses of bronchial epithelial cells to whole cigarette smoke mirror those of in-vivo exposed human bronchial mucosa.

BACKGROUND: Despite the well-known detrimental effects of cigarette smoke (CS), little is known about the complex gene expression dynamics in the early stages after exposure. This study aims to investigate early transcriptomic responses following CS exposure of airway epithelial cells in culture and compare these to those found in human CS exposure studies. METHODS: Primary bronchial epithelial cells (PBEC) were differentiated at the air-liquid interface (ALI) and exposed to whole CS. Bulk RNA-sequencing was performed at 1 h, 4 h, and 24 h hereafter, followed by differential gene expression analysis. Results were additionally compared to data retrieved from human CS studies. RESULTS: ALI-PBEC gene expression in response to CS was most significantly changed at 4 h after exposure. Early transcriptomic changes (1 h, 4 h post CS exposure) were related to oxidative stress, xenobiotic metabolism, higher expression of immediate early genes and pro-inflammatory pathways (i.e., Nrf2, AP-1, AhR). At 24 h, ferroptosis-associated genes were significantly increased, whereas PRKN, involved in removing dysfunctional mitochondria, was downregulated. Importantly, the transcriptome dynamics of the current study mirrored in-vivo human studies of acute CS exposure, chronic smokers, and inversely mirrored smoking cessation. CONCLUSION: These findings show that early after CS exposure xenobiotic metabolism and pro-inflammatory pathways were activated, followed by activation of the ferroptosis-related cell death pathway. Moreover, significant overlap between these transcriptomic responses in the in-vitro model and human in-vivo studies was found, with an early response of ciliated cells. These results provide validation for the use of ALI-PBEC cultures to study the human lung epithelial response to inhaled toxicants.

Organoid-based expansion of patient-derived primary alveolar type 2 cells for establishment of alveolus epithelial Lung-Chip cultures.

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Lung epithelial cells interact with immune cells and bacteria to shape the microenvironment in tuberculosis.

The lung epithelium has long been overlooked as a key player in tuberculosis disease. In addition to acting as a direct barrier to Mycobacterium tuberculosis (Mtb), epithelial cells (EC) of the airways and alveoli act as first responders during Mtb infections; they directly sense and respond to Mtb by producing mediators such as cytokines, chemokines and antimicrobials. Interactions of EC with innate and adaptive immune cells further shape the immune response against Mtb. These three essential components, epithelium, immune cells and Mtb, are rarely studied in conjunction, owing in part to difficulties in coculturing them. Recent advances in cell culture technologies offer the opportunity to model the lung microenvironment more closely. Herein, we discuss the interplay between lung EC, immune cells and Mtb and argue that modelling these interactions is of key importance to unravel early events during Mtb infection.

Dynamic differences in dietary polyunsaturated fatty acid metabolism in sputum of COPD patients and controls.

INTRODUCTION: Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be converted into lipid mediators here collectively named oxylipins. These include classical eicosanoids, but also pro-resolving mediators. A balanced production of pro-inflammatory and pro-resolving oxylipins is of importance for adequate inflammatory responses and subsequent return to homeostasis. OBJECTIVES: Here we investigated if PUFA metabolism is disturbed in COPD patients. METHODS: Free PUFA and oxylipin levels were measured in induced sputum samples from the Bergen COPD cohort and COPD exacerbation study using liquid chromatography-mass spectrometry. Additionally, effects of whole cigarette smoke on PUFA metabolism in air-liquid interface cultures of primary bronchial epithelial cells were assessed. RESULTS: Significantly lower levels of free alpha-linolenic acid, linoleic acid and eicosapentaenoic acid (EPA) were detected in sputum from stable COPD patients compared to controls. During acute exacerbation (AE), levels of free arachidonic acid and docosapentaenoic acid were higher than in stable COPD patients. Furthermore, levels of omega-3 EPA- and docosahexaenoic acid-derived oxylipins were lower in sputum from stable COPD patients compared to controls. Cyclooxygenase-2-converted mediators were mostly increased during AE. In vitro studies additionally showed that cigarette smoke exposure may also directly contribute to altered epithelial PUFA metabolism, and indirectly by causing airway epithelial remodelling. CONCLUSIONS: Our findings show significant differences in PUFA metabolism in COPD patients compared to controls, further changed during AE. Airway epithelial remodelling may contribute to these changes. These findings provide new insight in impaired inflammatory resolution in COPD.

Contribution of Host Defence Proteins and Peptides to Host-Microbiota Interactions in Chronic Inflammatory Lung Diseases.

The respiratory tract harbours a variety of microorganisms, collectively called the respiratory microbiota. Over the past few years, alterations in respiratory and gut microbiota composition have been associated with chronic inflammatory diseases of the lungs. How these changes influence disease development and progression is an active field of investigation. Identifying and understanding host-microbiota interactions and factors contributing to these interactions could promote the development of novel therapeutic strategies aimed at restoring host-microbiota homeostasis. In this review, we discuss recent literature on host-microbiota interactions in the respiratory tract, with a specific focus on the influence of endogenous host defence peptides and proteins (HDPs) on the composition of microbiota populations in vivo and explore possible HDPs-related therapeutic approaches targeting microbiota dysbiosis in chronic inflammatory lung diseases.

Immunomodulatory innate defence regulator (IDR) peptide alleviates airway inflammation and hyper-responsiveness.

BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections.

Cigarette smoke differentially affects IL-13-induced gene expression in human airway epithelial cells.

Allergic airways inflammation in asthma is characterized by an airway epithelial gene signature composed of POSTN, CLCA1, and SERPINB2 This Th2 gene signature is proposed as a tool to classify patients with asthma into Th2-high and Th2-low phenotypes. However, many asthmatics smoke and the effects of cigarette smoke exposure on the epithelial Th2 gene signature are largely unknown. Therefore, we investigated the combined effect of IL-13 and whole cigarette smoke (CS) on the Th2 gene signature and the mucin-related genes MUC5AC and SPDEF in air-liquid interface differentiated human bronchial (ALI-PBEC) and tracheal epithelial cells (ALI-PTEC). Cultures were exposed to IL-13 for 14 days followed by 5 days of IL-13 with CS exposure. Alternatively, cultures were exposed once daily to CS for 14 days, followed by 5 days CS with IL-13. POSTN, SERPINB2, and CLCA1 expression were measured 24 h after the last exposure to CS and IL-13. In both models POSTN, SERPINB2, and CLCA1 expression were increased by IL-13. CS markedly affected the IL-13-induced Th2 gene signature as indicated by a reduced POSTN, CLCA1, and MUC5AC expression in both models. In contrast, IL-13-induced SERPINB2 expression remained unaffected by CS, whereas SPDEF expression was additively increased. Importantly, cessation of CS exposure failed to restore IL-13-induced POSTN and CLCA1 expression. We show for the first time that CS differentially affects the IL-13-induced gene signature for Th2-high asthma. These findings provide novel insights into the interaction between Th2 inflammation and cigarette smoke that is important for asthma pathogenesis and biomarker-guided therapy in asthma.

Proinflammatory Cytokines Impair Vitamin D-Induced Host Defense in Cultured Airway Epithelial Cells.

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-α and IL-1ß (TNF-α/IL-1ß), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-α/IL-1ß treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1ß, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and vitamin D receptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-α/IL-1ß-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor-mitogen-activated protein kinase pathway. These findings indicate that TNF-α/IL-1ß decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.

Assessing large-vessel endothelial permeability using near-infrared fluorescence imaging--brief report.

OBJECTIVE: Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation. APPROACH AND RESULTS: To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E-deficient mice by Western diet for 4 weeks. Evans blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evans blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction-absorbance methodology for Evans blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α-stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evans blue dye uptake in (apolipoprotein E-deficient) mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry. CONCLUSIONS: The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.

Cathelicidin LL-37 induces time-resolved release of LTB4 and TXA2 by human macrophages and triggers eicosanoid generation in vivo.

In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.

Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages.

LL-37/hCAP-18 is the only human member of the cathelicidin family and plays an important role in killing various pathogens, as well as in immune modulation. In this study, we investigated the effect of LL-37 on bacterial phagocytosis by macrophages and demonstrate that LL-37 enhances phagocytosis of IgG-opsonized Gram-negative and Gram-positive bacteria in a dose- and time-dependent manner by dTHP-1 cells. In addition, LL-37 enhanced phagocytosis of nonopsonized Escherichia coli by human macrophages. Consistently, LL-37 elevated the expression of FcγRs on macrophages but not the complement receptors CD11b and -c. Further studies revealed that the expression of TLR4 and CD14 is also increased on LL-37-treated macrophages. Several lines of evidence indicated that the FPR2/ALX receptor mediated LL-37-induced phagocytosis. However, TLR4 signaling was also coupled to the phagocytic response, as a specific TLR4 antibody significantly suppressed phagocytosis of IgG-opsonized E. coli and nonopsonized E. coli by dTHP-1 cells. Finally, macrophages from Cnlp(-/-) mice exhibited diminished bacterial phagocytosis compared with macrophages from their WT littermates. In conclusion, we demonstrate a novel, immune-modulatory mechanism of LL-37, which may contribute to bacterial clearance.

The human lactoferrin-derived peptide hLF1-11 exerts immunomodulatory effects by specific inhibition of myeloperoxidase activity.

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.