The Novel Inducer of Innate Immunity HO53 Stimulates Autophagy in Human Airway Epithelial Cells.

Aroylated phenylenediamines (APDs) are novel modulators of innate immunity with respect to enhancing the expression of antimicrobial peptides and maintaining epithelial barrier integrity. Here, we present a new study on induction of autophagy in human lung epithelial cells by the APD HO53. Interestingly, HO53 affected autophagy in a dose-dependent manner, demonstrated by increased microtubule-associated proteins 1A/1B light-chain 3B (LC3B) processing in mature polarized bronchial epithelial cells. The quantification of LC3B puncta showed increased autophagy flux and formation of autophagosomes visualized by transmission electron microscopy. The phenotypic changes indicated that autophagy induction was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK), nuclear translocation of transcription factor EB (TFEB), and changes in expression of autophagy-related genes. The kinetics of the explored signaling pathways indicated on activation of AMPK followed by the nuclear translocation of TFEB. Moreover, our data suggest that HO53 modulates epigenetic changes related to induction of autophagy manifested by transcriptional regulation of histone-modifying enzymes. These changes were reflected by decreased ubiquitination of histone 2B at the lysine 120 residue that is associated with autophagy induction. Taken together, HO53 modulates autophagy, a part of the host defense system, through a complex mechanism involving several pathways and epigenetic events.

Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro.

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.

Gut microbiota-CRAMP axis shapes intestinal barrier function and immune responses in dietary gluten-induced enteropathy.

In the gut, cathelicidin-related antimicrobial peptide (CRAMP) has been largely described for its anti-infective activities. With an increasing recognition of its immune regulatory effects in extra-intestinal diseases, the role of CRAMP in gluten-induced small intestinal enteropathy celiac disease remains unknown. This study aimed to investigate the unexplored role of CRAMP in celiac disease. By applying a mouse model of gluten-induced enteropathy (GIE) recapitulating small intestinal enteropathy of celiac disease, we observed defective CRAMP production in duodenal epithelium during GIE. CRAMP-deficient mice were susceptible to the development of GIE. Exogenous CRAMP corrected gliadin-triggered epithelial dysfunction and promoted regulatory immune responses at the intestinal mucosa. Additionally, GIE-associated gut dysbiosis with enriched Pseudomonas aeruginosa and production of the protease LasB contributed to defective intestinal CRAMP production. These results highlight microbiota-CRAMP axis in the modulation of barrier function and immune responses in GIE. Hence, modulating CRAMP may represent a therapeutic strategy for celiac disease.

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity.

BACKGROUND: Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. METHODS: A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, ß-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). RESULTS: VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. CONCLUSIONS: Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.

Slow radiological improvement and persistent low-grade inflammation after chemotherapy in tuberculosis patients with type 2 diabetes.

BACKGROUND: Diabetes mellitus type 2 (DM) may impede immune responses in tuberculosis (TB) and thus contribute to enhanced disease severity. In this study, we aimed to evaluate DM-mediated alterations in clinical, radiological and immunological outcomes in TB disease. METHODS: Newly diagnosed pulmonary TB patients with or without DM (TB n = 40; TB-DM n = 40) were recruited in Dhaka, Bangladesh. Clinical symptoms, sputum smear and culture conversion as well as chest radiography were assessed. Peripheral blood and sputum samples were collected at the time of diagnosis (baseline) and after 1, 2 and 6 months of standard anti-TB treatment. Blood samples were also obtained from healthy controls (n = 20). mRNA expression of inflammatory markers in blood and sputum samples were quantified using real-time PCR. RESULTS: The majority of TB-DM patients had poor glycemic control (HbA1c > 8%) and displayed elevated pulmonary pathology (P = 0.039) particularly in the middle (P < 0.004) and lower lung zones (P < 0.02) throughout the treatment period. However, reduction of clinical symptoms and time to sputum smear and culture conversion did not differ between the groups. Transcripts levels of the pro-inflammatory cytokines IL-1ß (P = 0.003 at month-1 and P = 0.045 at month-2) and TNF-α (P = 0.005 at month-1) and the anti-inflammatory cytokine IL-10 (P = 0.005 at month-2) were higher in peripheral blood after anti-TB treatment in TB-DM compared to TB patients. Conversely in sputum, TB-DM patients had reduced CD4 (P < 0.009 at month-1) and IL-10 (P = 0.005 at month-1 and P = 0.006 at month-2) transcripts, whereas CD8 was elevated (P = 0.016 at month-2). At 1- and 2-month post-treatment, sputum IL-10 transcripts were inversely correlated with fasting blood glucose and HbA1c levels in all patients. CONCLUSION: Insufficient up-regulation of IL-10 in the lung may fuel persistent local inflammation thereby promoting lung pathology in TB-DM patients with poorly controlled DM.

Innate Effector Systems in Primary Human Macrophages Sensitize Multidrug-Resistant Klebsiella pneumoniae to Antibiotics.

Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are difficult to treat with conventional antibiotics. Thus, alternative strategies to control the growth of MDR Klebsiella are warranted. We hypothesized that activation of innate effector systems could sensitize MDR K. pneumoniae to conventional antibiotics. Thus, human primary macrophages were stimulated with compounds known to activate innate immunity (vitamin D3, phenylbutyrate [PBA], and the aroylated phenylenediamine HO53) and then infected with MDR Klebsiella in the presence or absence of antibiotics. Antibiotics alone were ineffective against MDR Klebsiella in the cellular model, whereas vitamin D3, PBA, and HO53 reduced intracellular growth by up to 70%. The effect was further improved when the innate activators were combined with antibiotics. Vitamin D3- and PBA-induced bacterial killing was dependent on CAMP gene expression, whereas HO53 needed the production of reactive oxygen species (ROS), as shown in cells where the CYBB gene was silenced and in cells from a patient with reduced ROS production due to a deletion in the CYBB gene and skewed lyonization. The combination of innate effector activation by vitamin D3, PBA, and HO53 was effective in sensitizing MDR Klebsiella to conventional antibiotics in a primary human macrophage model. This study provides new evidence for future treatment options for K. pneumoniae.

The anti-microbial peptide LL-37/CRAMP levels are associated with acute heart failure and can attenuate cardiac dysfunction in multiple preclinical models of heart failure.

Rationale: Biomarkers for the diagnosis of heart failure (HF) are clinically essential. Circulating antimicrobial peptides LL-37 has emerged as a novel biomarker in cardiovascular disease, however, its relevance as a biomarker for acute HF are undetermined. Methods: Acute HF patients were enrolled in this study and the serum levels of LL-37/CRAMP (cathelicidin-related antimicrobial peptide) were measured by ELISA. The receiver-operator characteristic (ROC) curve was used to determine if serum LL-37 could be a biomarker for acute HF. Mouse CRAMP (mCRAMP, mouse homolog for human LL-37) was also determined in both heart and serum samples of, transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced HF mice models, and phenylephrine (PE) and angiotensin II (AngII)-induced neonatal mouse cardiomyocytes (NMCMs) hypertrophic models, both intracellular and secreted, by ELISA. The protective effects of mCRAMP were determined in TAC, ISO, and AngII-induced HF in mice while whether HF was exacerbated in AngII-infused animals were checked in mCRAMP knockout mice. The underlying mechanism for protective effects of CARMP in pathological hypertrophy was determined by using a NF-κB agonist together with rCRAMP (rat homolog for human LL-37) in AngII or PE treated neonatal rat cardiomyocytes (NRCMs). Results: Serum levels of LL-37 were significantly decreased in acute HF patients (area under the curve (AUC) of 0.616), and negatively correlated with NT-proBNP. We further confirmed that mCRAMP was decreased in both heart and serum samples of TAC- and ISO-induced HF mice models. Moreover, in PE and AngII-induced NMCMs hypertrophic models, both intracellular and secreted mCRAMP levels were reduced. Functionally, mCRAMP could attenuate TAC, ISO, and AngII-induced HF in mice while CRAMP deficiency exacerbated HF. Mechanistically, the anti-hypertrophy effects of CRAMP were mediated by NF-κB signaling. Conclusions: Collectively, serum LL-37 is associated with acute HF and increasing CRAMP is protective against deleterious NF-κB signaling in the rodent.

Host Directed Therapy Against Infection by Boosting Innate Immunity.

The innate immune system constitutes the first line of defense against invading pathogens, regulating the normal microbiota and contributes to homeostasis. Today we have obtained detailed knowledge on receptors, signaling pathways, and effector molecules of innate immunity. Our research constellation has focused on ways to induce the expression of antimicrobial peptides (AMPs), the production of oxygen species (ROS and NO), and to activate autophagy, during the last two decades. These innate effectors, with different mechanisms of action, constitute a powerful defense armament in phagocytes and in epithelial cells. Innate immunity does not only protect the host from invading pathogens, but also regulates the composition of the microbiota, which is an area of intense research. Notably, some virulent bacteria have the capacity to downregulate innate defenses and can thereby cause invasive disease. Understanding the detailed mechanisms behind pathogen-mediated suppression of innate effectors are currently in progress. This information can be of importance for the development of novel treatments based on counteraction of the downregulation; we have designated this type of treatment as host directed therapy (HDT). The concept to boost innate immunity may be particularly relevant as many pathogens are developing resistance against classical antibiotics. Many pathogens that are resistant to antibiotics are sensitive to the endogenous effectors included in early host defenses, which contain multiple effectors working in cooperation to control infections. Here, we review recent data related to downregulation of AMPs by pathogenic bacteria, induction of innate effector mechanisms, including cytokine-mediated effects, repurposed drugs and the role of antibiotics as direct modulators of host responses. These findings can form a platform for the development of novel treatment strategies against infection and/or inflammation.

Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Cathelicidin-related antimicrobial peptide protects against ischaemia reperfusion-induced acute kidney injury in mice.

BACKGROUND AND PURPOSE: Despite recent advances in understanding its pathophysiology, treatment of acute kidney injury (AKI) remains a major unmet medical need, and novel therapeutic strategies are needed. Cathelicidin-related antimicrobial peptide (CRAMP) with immunomodulatory properties has an emerging role in various disease contexts. Here, we aimed to investigate the role of CRAMP and its underlying mechanisms in AKI. EXPERIMENTAL APPROACH: The human homologue LL-37 and CRAMP were measured in blood samples of AKI patients and in experimental AKI mice respectively. Experimental AKI was induced in wild-type and CRAMP-deficient (Cnlp-/- ) mice by ischaemia/reperfusion (I/R). Therapeutic evaluation of CRAMP was performed with exogenous CRAMP (5 mg·kg-1 , i.p.) treatment. KEY RESULTS: Cathelicidin expression was inversely related to clinical signs in patients and down-regulated in renal I/R-induced injury in mice. Cnlp-/- mice exhibited exacerbated I/R-induced renal dysfunction, aggravated inflammatory responses and apoptosis. Moreover, over-activation of the NLRP3 inflammasome in Cnlp-/- mice was associated with I/R-induced renal injury. Exogenous CRAMP treatment markedly attenuated I/R-induced renal dysfunction, inflammatory response and apoptosis, correlated with modulation of immune cell infiltration and phenotype. Consistent with Cnlp-/- mouse data, CRAMP administration suppressed renal I/R-induced NLRP3 inflammasome activation, and its renal protective effects were mimicked by a specific NLRP3 inhibitor CY-09. The reno-protective and NLRP3 inhibitory effects of CRAMP required the EGF receptor. CONCLUSION AND IMPLICATIONS: Our results suggest that CRAMP acts as a novel immunomodulatory mediator of AKI and modulation of CRAMP may represent a potential therapeutic strategy.

Host-Directed Therapy as a Novel Treatment Strategy to Overcome Tuberculosis: Targeting Immune Modulation.

Tuberculosis (TB) is one of the leading causes of mortality and morbidity, particularly in developing countries, presenting a major threat to the public health. The currently recommended long term treatment regimen with multiple antibiotics is associated with poor patient compliance, which in turn, may contribute to the emergence of multi-drug resistant TB (MDR-TB). The low global treatment efficacy of MDR-TB has highlighted the necessity to develop novel treatment options. Host-directed therapy (HDT) together with current standard anti-TB treatments, has gained considerable interest, as HDT targets novel host immune mechanisms. These immune mechanisms would otherwise bypass the antibiotic bactericidal targets to kill Mycobacterium tuberculosis (Mtb), which may be mutated to cause antibiotic resistance. Additionally, host-directed therapies against TB have been shown to be associated with reduced lung pathology and improved disease outcome, most likely via the modulation of host immune responses. This review will provide an update of host-directed therapies and their mechanism(s) of action against Mycobacterium tuberculosis.

Cathelicidin Contributes to the Restriction of Leishmania in Human Host Macrophages.

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated-Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model.

Klebsiella pneumoniae Expressing VIM-1 Metallo-ß-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation.

Antibiotic-resistant Klebsiella pneumoniae isolates constitute a great clinical challenge. One important resistance mechanism in K. pneumoniae is the metallo-ß-lactamases (MBLs), which require zinc for their function. Thus, zinc chelation could be a strategy to resensitize K. pneumoniae to ß-lactams. However, the potential role for endogenous zinc chelators for this purpose remains to be explored. The aim was to search for endogenous factors that could resensitize MBL-expressing K. pneumoniae to cefotaxime (CTX). Clinical K. pneumoniae isolates expressing different MBLs were screened for sensitivity to CTX in supernatants from human HT-29 colonic epithelial cells. Factors influencing CTX susceptibility were isolated and identified with chromatographic and biochemical methods. Free zinc was measured with a Zinquin assay, the thiol content was assessed with a fluorometric thiol assay, and the reducing ability of the supernatant was measured with a fluorescent l-cystine probe. Urine samples from healthy volunteers were used to validate findings ex vivo VIM-1-expressing K. pneumoniae regained susceptibility to CTX when grown in supernatants from HT-29 cells. This effect was mediated via free thiols in the supernatant, including l-cysteine, and could be prevented by inhibiting thioredoxin reductase activity in the supernatant. Free thiols in urine samples appeared to have a similar function in restoring CTX activity against VIM-1-expressing K. pneumoniae in a zinc-dependent manner. We have identified l-cysteine as an endogenous zinc chelator resulting in the resensitization of VIM-1-expressing K. pneumoniae to CTX. These results suggest that natural zinc chelators in combination with conventional antibiotics could be used to treat infections caused by VIM-1-expressing pathogens.

Low Methoxyl Pectin Protects against Autoimmune Diabetes and Associated Caecal Dysfunction.

SCOPE: This study aims to examine the protective effects of specific low-methoxyl pectin (LMP) on the development of type 1 diabetes (T1D). METHODS AND RESULTS: Female non-obese diabetic (NOD) mice are weaned onto either control or 5% LMP supplemented diets for up to 22 weeks of age. T1D incidence, gut barrier function, and pancreatic-gut immune responses are analyzed. LMP supplementation significantly dampened the onset of T1D in NOD mice. LMP supplementation induces caecal homeostasis, as indicated by the increasing SCFAs production, higher expression of tight junction proteins claudin 1, zonula occludens-2 in caecum. Furthermore, LMP-mediated caecal homeostasis impacts gut-pancreatic immunity, as evidenced by increased regulatory T cell population, modulated inflammatory cytokine expression, and suppressed NOD like receptor protein 3 (NLRP3) inflammasome activation in both caecum and pancreas. CONCLUSION: The data demonstrate that LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment, providing a scientific basis for using LMP as a novel functional supplementation to intervene T1D.

Modulation of Gut Microbiota by Low Methoxyl Pectin Attenuates Type 1 Diabetes in Non-obese Diabetic Mice.

Intestinal homeostasis underpins the development of type 1 diabetes (T1D), and dietary manipulations to enhance intestinal homeostasis have been proposed to prevent T1D. The current study aimed to investigate the efficacy of supplementing a novel specific low-methoxyl pectin (LMP) dietary fiber in preventing T1D development. Female NOD mice were weaned onto control or 5% (wt/wt) LMP supplemented diets for up to 40 weeks of age, overt diabetes incidence and blood glucose were monitored. Then broad-spectrum antibiotics (ABX) treatment per os for 7 days followed by gut microbiota transfer was performed to demonstrate gut microbiota-dependent effects. Next-generation sequencing was used for analyzing the composition of microbiota in caecum. Concentration of short chain fatty acids were determined by GC-MS. The barrier reinforcing tight junction proteins zonula occludens-2 (ZO-2), claudin-1 and NOD like receptor protein 3 (NLRP3) inflammasome activation were determined by Western blot. The proportion of CD25+Foxp3+CD4+ regulatory T cell (Foxp3+ Treg) in the pancreas, pancreatic and mesenteric lymph nodes was analyzed by flow cytometry. We found that LMP supplementation ameliorated T1D development in non-obese diabetic (NOD) mice, as evidenced by decreasing diabetes incidence and fasting glucose levels in LMP fed NOD mice. Further microbiota analysis revealed that LMP supplementation prevented T1D-associated caecal dysbiosis and selectively enriched caecal bacterial species to produce more SCFAs. The LMP-mediated microbial balance further enhanced caecal barrier function and shaped gut-pancreatic immune environment, as characterized by higher expression of tight junction proteins claudin-1, ZO-2 in caecum, increased Foxp3+ Treg population and decreased NLRP3 inflammasome activation in both caecum and pancreas. The microbiota-dependent beneficial effect of LMP on T1D was further proven by the fact that aberration of caecal microbiota by ABX treatment worsened T1D autoimmunity and could be restored with transfer of feces of LMP-fed NOD mice. These data demonstrate that this novel LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment. This finding opens a realistic option for gut microbiota manipulation and prevention of T1D in humans.

Novel aroylated phenylenediamine compounds enhance antimicrobial defense and maintain airway epithelial barrier integrity.

Aroylated phenylenediamines (APDs) are novel inducers of innate immunity enhancing cathelicidin gene expression in human bronchial epithelial cell lines. Here we present two newly developed APDs and aimed at defining the response and signaling pathways for these compounds with reference to innate immunity and antimicrobial peptide (AMP) expression. Induction was initially defined with respect to dose and time and compared with the APD Entinostat (MS-275). The induction applies to several innate immunity effectors, indicating that APDs trigger a broad spectrum of antimicrobial responses. The bactericidal effect was shown in an infection model against Pseudomonas aeruginosa by estimating bacteria entering cells. Treatment with a selected APD counteracted Pseudomonas mediated disruption of epithelial integrity. This double action by inducing AMPs and enhancing epithelial integrity for one APD compound is unique and taken as a positive indication for host directed therapy (HDT). The APD effects are mediated through Signal transducer and activator of transcription 3 (STAT3) activation. Utilization of induced innate immunity to fight infections can reduce antibiotic usage, might be effective against multidrug resistant bacteria and is in line with improved stewardship in healthcare.

Cathelicidin-related antimicrobial peptide protects against myocardial ischemia/reperfusion injury.

BACKGROUND: Cathelicidins are a major group of natural antimicrobial peptides which play essential roles in regulating host defense and immunity. In addition to the antimicrobial and immunomodulatory activities, recent studies have reported the involvement of cathelicidins in cardiovascular diseases by regulating inflammatory response and microvascular dysfunction. However, the role of cathelicidins in myocardial apoptosis upon cardiac ischemia/reperfusion (I/R) injury remains largely unknown. METHODS: CRAMP (cathelicidin-related antimicrobial peptide) levels were measured in the heart and serum from I/R mice and in neonatal mouse cardiomyocytes treated with oxygen glucose deprivation/reperfusion (OGDR). Human serum cathelicidin antimicrobial peptide (LL-37) levels were measured in myocardial infarction (MI) patients. The role of CRAMP in myocardial apoptosis upon I/R injury was investigated in mice injected with the CRAMP peptide and in CRAMP knockout (KO) mice, as well as in OGDR-treated cardiomyocytes. RESULTS: We observed reduced CRAMP level in both heart and serum samples from I/R mice and in OGDR-treated cardiomyocytes, as well as reduced LL-37 level in MI patients. Knockdown of CRAMP enhanced cardiomyocyte apoptosis, and CRAMP KO mice displayed increased infarct size and myocardial apoptosis. In contrast, the CRAMP peptide reduced cardiomyocyte apoptosis and I/R injury. The CRAMP peptide inhibited cardiomyocyte apoptosis by activation of Akt and ERK1/2 and phosphorylation and nuclear export of FoxO3a. c-Jun was identified as a negative regulator of the CRAMP gene. Moreover, lower level of serum LL-37/neutrophil ratio was associated with readmission and/or death in MI patients during 1-year follow-up. CONCLUSIONS: CRAMP protects against cardiomyocyte apoptosis and cardiac I/R injury via activation of Akt and ERK and phosphorylation and nuclear export of FoxO3a. Increasing LL-37 might be a novel therapy for cardiac ischemic injury.

Innate lymphoid cell type 3-derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB.

Escherichia coli and Klebsiella pneumoniae are opportunistic pathogens that are commonly associated with infections at mucosal surfaces, such as the lung or the gut. The host response against these types of infections includes the release of epithelial-derived antimicrobial factors such as lipocalin-2 (LCN-2), a protein that specifically inhibits the iron acquisition of Enterobacteriaceae by binding and neutralizing the bacterial iron-scavenging molecule enterobactin. Regulation of epithelial antimicrobial responses, including the release of LCN-2, has previously been shown to depend on IL-22, a cytokine produced by innate lymphoid cells type 3 (ILC3) during Enterobacteriaceae infections. However, much remains unknown about the extent to which antimicrobial responses are regulated by IL-22 and how IL-22 regulates the expression and production of LCN-2 in intestinal epithelial cells (IECs). Our study demonstrates how IL-22-induced activation of STAT3 synergizes with NF-κB-activating cytokines to enhance LCN-2 expression in human IECs and elucidates how ILC3 are involved in LCN-2-mediated host defense against Enterobacteriaceae. Together, these results provide new insight into the role of ILC3 in regulating LCN-2 expression in human IECs and could prove useful in future studies aimed at understanding the host response against Enterobacteriaceae as well as for the development of antimicrobial therapies against Enterobacteriaceae-related infections.

A novel cysteine-linked antibacterial surface coating significantly inhibits bacterial colonization of nasal silicone prongs in a phase one pre-clinical trial.

Ventilator associated pneumonia and sepsis are frequent complications in neonatal care. Bacterial colonization of medical devices and interfaces used for respiratory support may contribute by functioning as a bacterial reservoir seeding bacteria into airways. We have developed an antibacterial surface coating based on a cysteine ligand covalently coupled via a spacer to a carboxylic backbone layer on an acrylic acid grafted silicone surface. This coating was applied on a commercially available nasal prong and the antibacterial effect was evaluated both in vitro and in vivo in a first-in-human phase 1 trial. The coated nasal prongs had strong antibacterial activity against both Gram-negative and Gram-positive bacteria in vitro. In a randomized pre-clinical trial study of 24 + 24 healthy adult volunteers who carried coated or non-coated nasal prongs for 18 h, a 10log difference in mean bacterial colonization of 5.82 (p < 0.0001) was observed. These results show that this coating technique can prevent colonization by the normal skin and mucosal flora, and thus represent a promising novel technology for reduction of medical device-associated hospital acquired infections.

Immune responses in the treatment of drug-sensitive pulmonary tuberculosis with phenylbutyrate and vitamin D3 as host directed therapy.

BACKGROUND: We have previously shown that 8 weeks' treatment with phenylbutyrate (PBA) (500mgx2/day) with or without vitamin D3 (vitD3) (5000 IU/day) as host-directed therapy (HDT) accelerated clinical recovery, sputum culture conversion and increased expression of cathelicidin LL-37 by immune cells in a randomized, placebo-controlled trial in adults with pulmonary tuberculosis (TB). In this study we further aimed to examine whether HDT with PBA and vitD3 promoted clinically beneficial immunomodulation to improve treatment outcomes in TB patients. METHODS: Cytokine concentration was measured in supernatants of peripheral blood mononuclear cells (PBMC) from patients (n = 31/group). Endoplasmic reticulum stress-related genes (GADD34 and XBP1spl) and human beta-defensin-1 (HBD1) gene expression were studied in monocyte-derived-macrophages (MDM) (n = 18/group) from PBMC of patients. Autophagy in MDM (n = 6/group) was evaluated using LC3 expression by confocal microscopy. RESULTS: A significant decline in the concentration of cytokines/chemokines was noted from week 0 to 8 in the PBA-group [TNF-α (ß = - 0.34, 95% CI = - 0.68, - 0.003; p = 0.04), CCL11 (ß = - 0.19, 95% CI = - 0.36, - 0.03; p = 0.02) and CCL5 (ß = - 0.08, 95% CI = - 0.16, 0.002; p = 0.05)] and vitD3-group [(CCL11 (ß = - 0.17, 95% CI = - 0.34, - 0.001; p = 0.04), CXCL10 (ß = - 0.38, 95% CI = - 0.77, 0.003; p = 0.05) and PDGF-ß (ß = - 0.16, 95% CI = - 0.31, 0.002; p = 0.05)] compared to placebo. Both PBA- and vitD3-groups showed a decline in XBP1spl mRNA on week 8 (p < 0.03). All treatment groups demonstrated increased LC3 expression in MDM compared to placebo over time (p < 0.037). CONCLUSION: The use of PBA and vitD3 as adjunct therapy to standard TB treatment promoted favorable immunomodulation to improve treatment outcomes. TRIALS REGISTRATION: This trial was retrospectively registered in clinicaltrials.gov, under identifier NCT01580007 .