Butyrate induces STAT3/HIF-1α/IL-22 signaling via GPCR and HDAC3 inhibition to activate autophagy in head kidney macrophages from turbot (Scophthalmus maximus L.).

As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages.

Butyrate-induced IL-22 expression in fish macrophages contributes to bacterial clearance.

IL-22 has been characterized as a critical cytokine in maintaining barrier integrity and host immunity. So far, it has been known that IL-22 is mainly produced by lymphoid lineage cells. In the present study, we have thoroughly investigated butyrate-induced production and function of IL-22 in fish macrophages. Our results demonstrated that short-chain fatty acids (SCFAs), major microbiota-derived metabolites, promoted the expression of IL-22 in head kidney macrophages (HKMs) of turbot (Scophthalmus maximus L.). Interestingly, butyrate-mediated intracellular bacterial killing in HKMs diminished when IL-22 expression was interfered. Furthermore, the turbot fed the diet containing sodium butyrate (NaB) exhibited significantly lower mortality after bacterial infection, compared to the fish fed a basal diet. At the meantime, a higher level of IL-22 expression and bactericidal activity was detected in HKMs from the turbot fed NaB-supplemented diet. In addition, NaB treatment promoted the expression of antimicrobial peptides (AMPs) ß-defensins in zebrafish (Danio rerio). However, butyrate-induced expression of AMPs was reduced in IL-22 mutant zebrafish compared to wild-type (WT) fish. Meanwhile, NaB treatment was incapable to protect IL-22 mutant fish from bacterial infection as it did in WT zebrafish. Importantly, our results demonstrated that IL-22 expression was remarkably suppressed in macrophage-depleted zebrafish, indicating that macrophage might be a cell source of IL-22 production in vivo. In conclusion, all these findings collectively revealed that SCFAs regulated the production and function of IL-22 in fish macrophages, which facilitated host resistance to bacterial invasion.

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.

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.

Azithromycin has lung barrier protective effects in a cell model mimicking ventilator-induced lung injury.

Azithromycin (AZM) is a broad-spectrum antibiotic widely used to treat infections. AZM also has been shown to have anti-inflammatory and immunomodulatory functions unrelated to its antibacterial activity that contribute to the effectiveness of this drug in chronic respiratory diseases. The mechanisms behind these beneficial effects are not yet fully elucidated. We have previously shown that AZM enhances barrier integrity of bronchial epithelial cells and directs them towards epidermal differentiation. In this study, we analyzed the effect of AZM pre-treatment of human bronchial and alveolar derived cell lines on mechanical stress in a cyclical pressure air-liquid interface device (CPAD) that models the disruption of the epithelial barrier with increased inflammatory response in lung tissue, which is associated with ventilator-induced lung injury (VILI). Immunostaining and electron microscopy showed that barrier integrity of the epithelium was compromised by cyclically stressing the cells but maintained when cells had been pre-treated with AZM. Lamellar body formation was revealed in AZM pre-treated cells, possibly further supporting the barrier-enhancing effects. RNA sequencing showed that the inflammatory response was attenuated by AZM treatment before cyclical stress. YKL-40, an emerging inflammatory marker, increased both due to cyclical stress and upon AZM treatment. These data confirm the usefulness of the CPAD to model ventilator-induced lung injury and suggest that AZM has barrier protective and immunomodulatory effects, attenuating the inflammatory response during mechanical stress, and might therefore be lung protective during mechanical ventilation. The model could be used to assess further drug candidates that influence barrier integrity and modulate inflammatory response.

Innovative in vitro method to study ventilator induced lung injury.

Mechanical ventilation (MV) is a life-saving therapy for critically ill patients, alleviating the work of breathing and supporting adequate gas exchange. However, MV can cause ventilator induced lung injury (VILI) by baro/volu- and atelectrauma, even lead to acute respiratory distress syndrome (ARDS), and substantially augment mortality. There is a need for specific biomarkers and novel research platforms for VILI/ARDS research to study these detrimental disorders and seek ways to avoid or prevent them. Previous in vitro studies on bronchial epithelium, cultured in air-liquid interface (ALI) conditions, have generally utilized static or constant pressure.  We have developed a Cyclical Pressure ALI Device (CPAD) that enables cyclical stress on ALI cultured human bronchial cells, with the aim of mimicking the effects of MV. Using CPAD we were able to analyze differentially expressed VILI/ARDS and innate immunity associated genes along with increased expression of associated proteins. CPAD provides an easy and accessible way to analyze functional and phenotypic changes that occur during VILI and may provide a platform for future drug testing.

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.

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 .

Lactose Induces Phenotypic and Functional Changes of Neutrophils and Macrophages to Alleviate Acute Pancreatitis in Mice.

Acute pancreatitis (AP) is one common clinical acute abdominal disease, for which specific pharmacological or nutritional therapies remain elusive. Lactose, a macronutrient and an inducer of host innate immune responses, possesses immune modulatory functions. The current study aimed to investigate potential modulatory effects of lactose and the interplay between the nutrient and pancreatic immunity during experimentally induced AP in mice. We found that either prophylactic or therapeutic treatment of lactose time-dependently reduced the severity of AP, as evidenced by reduced pancreatic edema, serum amylase levels, and pancreatic myeloperoxidase activities, as well as by histological examination of pancreatic damage. Overall, lactose promoted a regulatory cytokine milieu in the pancreas and reduced infiltration of inflammatory neutrophils and macrophages. On acinar cells, lactose was able to suppress caerulein-induced inflammatory signaling pathways and to suppress chemoattractant tumor necrosis factor (TNF)-α and monocyte chemotactic protein-1 production. Additionally, lactose acted on pancreas-infiltrated macrophages, increasing interleukin-10 and decreasing tumor necrosis factor alpha production. Notably, lactose treatment reversed AP-associated infiltration of activated neutrophils. Last, the effect of lactose on neutrophil infiltration was mimicked by a galectin-3 antagonist, suggesting a potential endogenous target of lactose. Together, the current study demonstrates an immune regulatory effect of lactose to alleviate AP and suggests its potential as a convenient, value-added therapeutic macronutrient to control AP, and lower the risk of its systemic complications.

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors.

Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human ß-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.

Glucocorticoid dexamethasone down-regulates basal and vitamin D3 induced cathelicidin expression in human monocytes and bronchial epithelial cell line.

Glucocorticoids (GCs) have been extensively used as the mainstream treatment for chronic inflammatory disorders. The persistent use of steroids in the past decades and the association with secondary infections warrants for detailed investigation into their effects on the innate immune system and the therapeutic outcome. In this study, we analyse the effect of GCs on antimicrobial polypeptide (AMP) expression. We hypothesize that GC related side effects, including secondary infections are a result of compromised innate immune responses. Here, we show that treatment with dexamethasone (Dex) inhibits basal mRNA expression of the following AMPs; human cathelicidin, human beta defensin 1, lysozyme and secretory leukocyte peptidase 1 in the THP-1 monocytic cell-line (THP-1 monocytes). Furthermore, pre-treatment with Dex inhibits vitamin D3 induced cathelicidin expression in THP-1 monocytes, primary monocytes and in the human bronchial epithelial cell line BCi NS 1.1. We also demonstrate that treatment with the glucocorticoid receptor (GR) inhibitor RU486 counteracts Dex mediated down-regulation of basal and vitamin D3 induced cathelicidin expression in THP-1 monocytes. Moreover, we confirmed the anti-inflammatory effect of Dex. Pre-treatment with Dex inhibits dsRNA mimic poly IC induction of the inflammatory chemokine IP10 (CXCL10) and cytokine IL1B mRNA expression in THP-1 monocytes. These results suggest that GCs inhibit innate immune responses, in addition to exerting beneficial anti-inflammatory effects.

Cathelicidins positively regulate pancreatic ß-cell functions.

Cathelicidins are pleiotropic antimicrobial peptides largely described for innate antimicrobial defenses and, more recently, immunomodulation. They are shown to modulate a variety of immune or nonimmune host cell responses. However, how cathelicidins are expressed by ß cells and modulate ß-cell functions under steady-state or proinflammatory conditions are unknown. We find that cathelicidin-related antimicrobial peptide (CRAMP) is constitutively expressed by rat insulinoma ß-cell clone INS-1 832/13. CRAMP expression is inducible by butyrate or phenylbutyric acid and its secretion triggered upon inflammatory challenges by IL-1ß or LPS. CRAMP promotes ß-cell survival in vitro via the epidermal growth factor receptor (EGFR) and by modulating expression of antiapoptotic Bcl-2 family proteins: p-Bad, Bcl-2, and Bcl-xL. Also via EGFR, CRAMP stimulates glucose-stimulated insulin secretion ex vivo by rat islets. A similar effect is observed in diabetes-prone nonobese diabetic (NOD) mice. Additional investigation under inflammatory conditions reveals that CRAMP modulates inflammatory responses and ß-cell apoptosis, as measured by prostaglandin E2 production, cyclooxygenases (COXs), and caspase activation. Finally, CRAMP-deficient cnlp(-/-) mice exhibit defective insulin secretion, and administration of CRAMP to prediabetic NOD mice improves blood glucose clearance upon glucose challenge. Our finding suggests that cathelicidins positively regulate ß-cell functions and may be potentially used for intervening ß-cell dysfunction-associated diseases.

Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial.

BACKGROUND: Development of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects. OBJECTIVE: To examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients. METHODS: Adult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and ex vivo bactericidal function of monocyte-derived macrophages (MDM) were determined at week 0, 4, 8, 12 and additionally at week 24 for plasma 25(OH)D3. RESULTS: At week 4, 71% (46/65) of the patients in the PBA+vitD3-group (p = 0.001) and 61.3% (38/62) in the vitD3-group (p = 0.032) were culture negative compared to 42.2% (27/64) in the placebo-group. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (p = 0.001) and 2.2 times higher in vitD3-group (p = 0.032) compared to placebo. The concentration of LL-37 in MDM was significantly higher in the PBA-group compared to placebo at week 12 (p = 0.034). Decline in intracellular Mtb growth in MDM was earlier in the PBA-group compared to placebo (log rank 11.38, p = 0.01). CONCLUSION: Adjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB. TRIAL REGISTRATION: clinicaltrials.gov NCT01580007.

Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages.

LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca(2+), and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.

Boosting innate immunity: development and validation of a cell-based screening assay to identify LL-37 inducers.

Innate immunity, the front line of our defence against pathogens, relies, to a great extent, on the production of antimicrobial peptides (AMPs). These peptides exhibit antimicrobial activity and immunomodulatory properties. In humans, AMPs include the defensins (α- and ß-families) and the cathelicidin, LL-37. Bacterial resistance against antibiotics is a growing concern, and novel antimicrobial strategies are needed urgently. Hence, the concept of strengthening immune defences against infectious microbes by inducing AMP expression may represent novel or complementary pharmaceutical interventions in the treatment or prevention of infections. We have developed and validated a robust cell-based reporter assay for LL-37 expression, which serves as a marker for a healthy epithelial barrier. This reporter assay can be a powerful tool for high-throughput screenings. We first employed our assay to screen a panel of histone deacetylase inhibitors and derivatives, and then the Prestwick Chemical Library of Food and Drug Administration-approved compounds. After hit confirmation and independent validation in the parental cell line we identified five novel inducers of LL-37. This reporter assay will help to identify novel drug candidates for the treatment and prevention of infections. Importantly, the pattern of hits obtained may suggest cellular pathways and key mediators involved in the regulation of AMP expression.

Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculosis.

BACKGROUND: We earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM). METHODS: Healthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method. RESULTS: MDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05). CONCLUSION: The results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007).

Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant.

Phenylbutyrate induces antimicrobial peptide expression.

Antimicrobial peptides (AMPs) are important components of our first line of defense. Induction of AMPs such as LL-37 of the cathelicidin family might provide a novel approach in treating bacterial infections. In this study we identified 4-phenylbutyrate (PBA) as a novel inducer of AMP expression and investigated affected regulatory pathways. We treated various cell lines with PBA and assessed mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Cathelicidin AMP (CAMP) gene expression was found to be upregulated in all four cell lines tested. Additionally, we found that the beta-defensin 1 gene was upregulated in the lung epithelial cell line VA10 while being downregulated in the monocytic cell line U937. Further we found that PBA induced CAMP gene expression synergistically with 1,25-dihydroxyvitamin D(3) at both protein and mRNA levels. The general mechanism of induction of CAMP gene expression by PBA was found to be dependent on protein synthesis. Results from quantitative chromatin immunoprecipitation experiments challenge the common view that histone deacetylase inhibitors directly increase CAMP gene expression. Furthermore, we have demonstrated that inhibition of the mitogen-activated protein kinases MEK1/2 and c-Jun N-terminal kinase attenuate PBA-induced CAMP gene expression. Similarly, alpha-methylhydrocinnamate (ST7), an analogue of PBA, increases CAMP gene expression. Our findings contribute to understanding of the regulation of AMP expression and suggest that PBA and/or ST7 is a promising drug candidate for treatment of microbial infections by strengthening the epithelial antimicrobial barriers.