Combination of esomeprazole and pirfenidone enhances antifibrotic efficacy in vitro and in a mouse model of TGFß-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology. Currently, pirfenidone and nintedanib are the only FDA-approved drugs for the treatment of IPF and are now the standard of care. This is a significant step in slowing down the progression of the disease. However, the drugs are unable to stop or reverse established fibrosis. Several retrospective clinical studies indicate that proton pump inhibitors (PPIs; FDA-approved to treat gastroesophageal reflux) are associated with favorable outcomes in patients with IPF, and emerging preclinical studies report that PPIs possess antifibrotic activity. In this study, we evaluated the antifibrotic efficacy of the PPI esomeprazole when combined with pirfenidone in vitro and in vivo. In cell culture studies of IPF lung fibroblasts, we assessed the effect of the combination on several fibrosis-related biological processes including TGFß-induced cell proliferation, cell migration, cell contraction, and collagen production. In an in vivo study, we used mouse model of TGFß-induced lung fibrosis to evaluate the antifibrotic efficacy of esomeprazole/pirfenidone combination. We also performed computational studies to understand the molecular mechanisms by which esomeprazole and/or pirfenidone regulate lung fibrosis. We found that esomeprazole significantly enhanced the anti-proliferative effect of pirfenidone and favorably modulated TGFß-induced cell migration and contraction of collagen gels. We also found that the combination significantly suppressed collagen production in response to TGFß in comparison to pirfenidone monotherapy. In addition, our animal study demonstrated that the combination therapy effectively inhibited the differentiation of lung fibroblasts into alpha smooth muscle actin (αSMA)-expressing myofibroblasts to attenuate the progression of lung fibrosis. Finally, our bioinformatics study of cells treated with esomeprazole or pirfenidone revealed that the drugs target several extracellular matrix (ECM) related pathways with esomeprazole preferentially targeting collagen family members while pirfenidone targets the keratins. In conclusion, our cell biological, computational, and in vivo studies show that the PPI esomeprazole enhances the antifibrotic efficacy of pirfenidone through complementary molecular mechanisms. This data supports the initiation of prospective clinical studies aimed at repurposing PPIs for the treatment of IPF and other fibrotic lung diseases where pirfenidone is prescribed.

p38 MAPK Activity Is Required to Prevent Hyperactivation of NLRP3 Inflammasome.

Inflammation contributes to the pathogenesis and morbidity of wide spectrum of human diseases. The inflammatory response must be actively controlled to prevent bystander damage to tissues. Yet, the mechanisms controlling excessive inflammatory responses are poorly understood. NLRP3 inflammasome plays an important role in innate immune response to cellular infection or stress. Its activation must be tightly regulated because uncontrolled inflammasome activation is associated with a number of human diseases. p38 MAPK signaling plays an essential role in the regulation of inflammation. The role of p38 MAPK in inflammatory response associated with the expression of proinflammatory molecules is known. However, the anti-inflammatory functions of p38 MAPK are largely unknown. In this study, we show that pharmacologic inhibition or genetic deficiency of p38 MAPK leads to hyperactivation of NLRP3 inflammasome, resulting in enhanced Caspase 1 activation and IL-1ß and IL-18 production. The deficiency of p38 MAPK activity induced an increase of cytosolic Ca2+ and excessive mitochondrial Ca2+ uptake, leading to exacerbation of mitochondrial damage, which was associated with hyperactivation of NLRP3 inflammasome. In addition, mice with deficiency of p38 MAPK in granulocytes had evidence of in vivo hyperactivation of NLRP3 inflammasome and were more susceptible to LPS-induced sepsis compared with wild-type mice. Our results suggest that p38 MAPK negatively regulates NLRP3 inflammasome through control of Ca2+ mobilization. Hyperactivity of inflammasome in p38-deficient mice causes lung inflammation and increased susceptibility to septic shock.

Esomeprazole attenuates inflammatory and fibrotic response in lung cells through the MAPK/Nrf2/HO1 pathway.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is an orphan disease characterized by progressive loss of lung function resulting in shortness of breath and often death within 3-4 years of diagnosis. Repetitive lung injury in susceptible individuals is believed to promote chronic oxidative stress, inflammation, and uncontrolled collagen deposition. Several preclinical and retrospective clinical studies in IPF have reported beneficial outcomes associated with the use of proton pump inhibitors (PPIs) such as esomeprazole. Accordingly, we sought to investigate molecular mechanism(s) by which PPIs favorably regulate the disease process. METHODS: We stimulated oxidative stress, pro-inflammatory and profibrotic phenotypes in primary human lung epithelial cells and fibroblasts upon treatment with bleomycin or transforming growth factor ß (TGFß) and assessed the effect of a prototype PPI, esomeprazole, in regulating these processes. RESULTS: Our study shows that esomeprazole controls pro-inflammatory and profibrotic molecules through nuclear translocation of the transcription factor nuclear factor-like 2 (Nrf2) and induction of the cytoprotective molecule heme oxygenase 1 (HO1). Genetic deletion of Nrf2 or pharmacological inhibition of HO1 impaired esomeprazole-mediated regulation of proinflammatory and profibrotic molecules. Additional studies indicate that activation of Mitogen Activated Protein Kinase (MAPK) pathway is involved in the process. Our experimental data was corroborated by bioinformatics studies of an NIH chemical library which hosts gene expression profiles of IPF lung fibroblasts treated with over 20,000 compounds including esomeprazole. Intriguingly, we found 45 genes that are upregulated in IPF but downregulated by esomeprazole. Pathway analysis showed that these genes are enriched for profibrotic processes. Unbiased high throughput RNA-seq study supported antifibrotic effect of esomeprazole and revealed several novel targets. CONCLUSIONS: Taken together, PPIs may play antifibrotic role in IPF through direct regulation of the MAPK/Nrf2/HO1 pathway to favorably influence the disease process in IPF.

Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR.

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS-deficient P. aeruginosa but does not affect the survival of ExoS-containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP-ribosyltransferase domain. Inhibition of mTOR is caused by ExoS-mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.

Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor.

Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.

Influence of probiotic and yeast culture supplementation on selected biochemical and immunological parameters of growing lambs.

This study was carried out to evaluate the potential effects of 90 days-long dietary supple- mentation of probiotic and yeast culture on immunity condition of lambs. Fifteen Rahmani growing male lambs (about 5 months old and 23.21±2.75 kg body weight) were randomly allo- cated to three equal groups consisting of 5 animals each. The animals in the first group, served as a control (group C), were fed a basal diet without any supplementation. The lambs in the second and third group were fed the basal diet supplemented with probiotic (group Y) or yeast culture (group YC), respectively. The probiotic consisted of live yeast (Saccharomyces cerevisae) alone, while the yeast culture was composed of Saccharomyces cerevisiae and the media on which it was grown. In group Y and YC, each lamb was supplemented daily with 0.5 g and 7.0 g of live yeast and yeast culture, respectively. Blood samples were collected before feeding the supplements and then every 15 days until the day 90th. Total and differential leucocytic counts, total protein, albumin, IgA, IgG and IgM levels were measured in blood. There were insignificant (p>0.05) variations in the levels of total and differential leucocytic counts and total protein among the groups throughout the experiment. However, significant differences (p⟨0.05) were found in globulin, IgA, IgG and IgM in both (Y) and (YC) groups, but the effect of yeast culture seems to be better than that of the probiotic. In conclusions, the obtained results indicate that the tested probiotic and yeast culture improve the immunological status of lambs.

Autophagy in Pulmonary Innate Immunity.

Autophagy is a major intracellular digestion system that delivers cytoplasmic components for degradation and recycling. In this capacity, autophagy plays an important role in maintaining cellular homeostasis by mediating the degradation of cellular macromolecules and dysfunctional organelles and regeneration of nutrients for cell growth. Autophagy is important in innate immunity, as it is responsible for the clearance of various pathogens. Deficiency of intracellular autophagy can result in exaggerated activation of the inflammasome. The latter is an innate immune complex that senses diverse pathogen-associated or danger-associated molecular patterns and activates the expression of inflammatory cytokines. In autophagy-deficient cells, accumulation of damaged organelles, misfolded proteins, and reactive oxygen species contribute to inflammasome activation. The lung is continuously exposed to pathogens from the environment, rendering it vulnerable to infection. The lung innate immune cells act as a crucial initial barrier against the continuous threat from pathogens. In this review, we will summarize recent findings on the regulation of autophagy and its inter-action with innate immunity, focusing on the lung.

EJP18 peptide derived from the juxtamembrane domain of epidermal growth factor receptor represents a novel membrane-active cell-penetrating peptide.

Membrane-active peptides have been extensively studied to probe protein-membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, non-toxic concentrations the peptides outperformed the well-characterised CPP octaarginine in cellular delivery capacity for a fluorophore or proteins that were associated with the peptide covalently or via ionic interactions. EJP18 thus represents a novel membrane-active peptide that may be used as a naturally derived model for biophysical protein-membrane interactions or for delivery of cargo into cells for therapeutic or diagnostic applications.

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response.

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits. In parallel, we found that mice lacking Upf2 in the forebrain (Upf2 fb-KO mice) show impaired NMD, memory deficits, abnormal long-term potentiation (LTP), and social and communication deficits. Surprisingly, Upf2 fb-KO mice exhibit elevated expression of immune genes and brain inflammation. More importantly, treatment with two FDA-approved anti-inflammatory drugs reduced brain inflammation, restored LTP and long-term memory, and reversed social and communication deficits. Collectively, our findings indicate that impaired UPF2-dependent NMD leads to neurodevelopmental dysfunction and suggest that anti-inflammatory agents may prove effective for treatment of disorders with impaired NMD.

An autophagy-inducing and TLR-2 activating BCG vaccine induces a robust protection against tuberculosis in mice.

Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis due to M. tuberculosis (Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG85C5 induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger TH1 cytokines (IL-12, IL-1ß, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG85C5 also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG85C5 infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG85C5 induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG85C5 markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG85C5 induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.

Nicotine Modulates Growth Factors and MicroRNA to Promote Inflammatory and Fibrotic Processes.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that destroys the structure and function of the lungs. Risk factors include advanced age and genetic predisposition. However, tobacco use is the chief modifiable risk factor. The prevalence of tobacco use in IPF reaches up to 80%. Although tobacco smoke contains over 5000 chemicals, nicotine is a major component. Nicotine is a bioactive molecule that acts upon nicotinic acetylcholine receptors expressed on neuronal and non-neuronal cells including endothelial cells. Accordingly, it has a pleiotropic effect on cell proliferation and angiogenesis. The angiogenic effect is partly mediated by stimulation of growth factors including fibroblast, platelet-derived, and vascular endothelial growth factors. Nintedanib, a Food and Drug Administration-approved drug for IPF, works by inhibiting receptors for these growth factors, suggesting a pathobiologic role of the growth factors in IPF and a potential mechanism by which tobacco use may exacerbate the disease process; additionally, nicotine downregulates anti-inflammatory microRNAs (miRs) in lung cells. Here, we profiled the expression of miRs in lung tissues explanted from a lung injury model and examined the effect of nicotine on one of the identified miRs (miR-24) and its downstream targets. Our data show that miR-24 is downregulated during lung injury and is suppressed by nicotine. We also found that nicotine upregulates the expression of inflammatory cytokines targeted by miR-24. Finally, nicotine stimulated growth factors, fibroblast proliferation, collagen release, and expression of myofibroblast markers. Taken together, nicotine, alone or as a component of tobacco smoke, may accelerate the disease process in IPF through stimulation of growth factors and downregulation of anti-inflammatory miRs.

Anticancer therapy and lung injury: molecular mechanisms.

INTRODUCTION: Chemotherapy and radiation therapy are two mainstream strategies applied in the treatment of cancer that is not operable. Patients with hematological or solid tumor malignancies substantially benefit from chemotherapeutic drugs and/or ionizing radiation delivered to the site of malignancy. However, considerable adverse effects, including lung inflammation and fibrosis, are associated with the use of these treatment modalities. Areas covered: As we move toward the era of precision health, we are compelled to understand the molecular basis of chemoradiation-induced pathological lung remodeling and to develop effective treatment strategies that mitigate the development of chronic lung disease (i.e. fibrosis) in cancer patients. The review discusses chemotherapeutic agents that are reported to induce or associate with acute and/or chronic lung injury. Expert commentary: There is a need to molecularly understand how chemotherapeutic drugs induce or associate with respiratory toxicities and whether such characteristics are inherently related to their antitumor effect or are collateral. Once such mechanisms have been identified and/or fully characterized, they may be able to guide disease-management decisions including effective intervention strategies for the adverse effects. In the meantime, radiation oncologists should be judicious on the dose of radiation delivered to the lungs, the volume of lung irradiated, and concurrent use of chemotherapeutic drugs.

Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1ß release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. METHODS: NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca2+ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. RESULTS: NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca2+ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9-mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. CONCLUSIONS: Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

The E3 ubiquitin ligase STUB1 regulates autophagy and mitochondrial biogenesis by modulating TFEB activity.

TFEB is a master regulator for transcription of genes involved in autophagy, lysosome and mitochondrial biogenesis. Activity of TFEB is inhibited upon its phosphorylation. STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin proteasome pathway. Thus, the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.

Mesenchymal stem cells internalize Mycobacterium tuberculosis through scavenger receptors and restrict bacterial growth through autophagy.

Human mesenchymal stem cells (MSCs) express scavenger receptors that internalize lipids, including oxidized low-density lipoprotein (oxLDL). We report that MSCs phagocytose Mycobacterium tuberculosis (Mtb) through two types of scavenger receptors (SRs; MARCO and SR-B1), as blockade of the receptors with antibodies or siRNA knockdown decreased the uptake of Mtb. MSCs also expressed mannose receptor (MR) that was found to endocytose rhodamine-labeled mannosylated BSA (rMBSA), though the receptor was not involved in the uptake of Mtb. Dil-oxLDL and rMBSA taken up into MSC endosomes colocalized with Mtb phagosomes, thus suggesting that the latter were fusion competent. Phagocytosed Mtb did not replicate within MSCs, thus suggesting an intrinsic control of bacterial growth. Indeed, MSCs exhibited intrinsic autophagy, which was up-regulated after activation with rapamycin. SiRNA knockdown of autophagy initiator beclin-1 enhanced Mtb survival, whereas rapamycin-induced autophagy increased intracellular killing of Mtb. In addition, MSCs secreted nitric oxide after Mtb infection, and inhibition of NO by N(G)-monomethyl-L-arginine enhanced intracellular survival of Mtb. MSCs can be grown in large numbers in vitro, and autologous MSCs transfused into tuberculosis patients have been found to be safe and improve lung immunity. Thus, MSCs are novel phagocytic cells with a potential for immunotherapy in treating multidrug-resistant tuberculosis.

STUB1 regulates TFEB-induced autophagy-lysosome pathway.

TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin-proteasome pathway. Phosphorylated TFEB accumulated in STUB1-deficient cells and in tissues of STUB1-deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1-deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.

LRP1-Dependent BMPER Signaling Regulates Lipopolysaccharide-Induced Vascular Inflammation.

OBJECTIVE: Bacterial endotoxin (lipopolysaccharide)-mediated sepsis involves dysregulated systemic inflammation, which injures the lung and other organs, often fatally. Vascular endothelial cells act as both targets and mediators of lipopolysaccharide-induced inflammatory responses. Dysfunction of endothelium results in increases of proinflammatory cytokine production and permeability leakage. BMPER (bone morphogenetic protein-binding endothelial regulator), an extracellular modulator of bone morphogenetic protein signaling, has been identified as a vital component in chronic endothelial inflammatory responses and atherosclerosis. However, it is unclear whether BMPER also regulates inflammatory response in an acute setting such as sepsis. To address this question, we investigated the role of BMPER during lipopolysaccharide-induced acute lung injury. APPROACH AND RESULTS: Mice missing 1 allele of BMPER (BMPER+/- mice used in the place of BMPER-/- mice that die at birth) were used for lipopolysaccharide challenge. Lipopolysaccharide-induced pulmonary inflammation and injury was reduced in BMPER+/- mice as shown by several measures, including survival rate, infiltration of inflammatory cells, edema, and production of proinflammatory cytokines. Mechanistically, we have demonstrated that BMPER is required and sufficient for the activation of nuclear factor of activated T cells c1. This BMPER-induced nuclear factor of activated T cells activation is coordinated by multiple signaling pathways, including bone morphogenetic protein-independent low-density lipoprotein receptor-related protein 1-extracellular signal-regulated kinase activation, calcineurin signaling, and low-density lipoprotein receptor-related protein 1ß-mediated nuclear factor 45 nuclear export in response to BMPER treatment. CONCLUSIONS: We conclude that BMPER plays a pivotal role in pulmonary inflammatory response, which provides new therapeutic options against sepsis shock. The new signaling pathway initiated by BMPER/low-density lipoprotein receptor-related protein 1 axis broadens our understanding about BMPER's role in vascular homeostasis.

Antepartum Antibiotic Treatment Increases Offspring Susceptibility to Experimental Colitis: A Role of the Gut Microbiota.

BACKGROUND AND AIMS: Postnatal maturation of the immune system is largely driven by exposure to microbes, and thus the nature of intestinal colonization may be associated with development of childhood diseases that may persist into adulthood. We investigated whether antepartum antibiotic (ATB) therapy can increase offspring susceptibility to experimental colitis through alteration of the gut microbiota. METHODS: Pregnant C57Bl/6 mice were treated with cefazolin at 160 mg/kg body weight or with saline starting six days before due date. At 7 weeks, fecal samples were collected from male offspring after which they received 4% dextran sulfate sodium (DSS) in drinking water for 5 days. Disease activity index, histology, colonic IL-6, IL-1ß and serum C-reactive protein (CRP) were determined. The V3-V4 region of colonic and fecal bacterial 16S rRNA was sequenced. Alpha-, beta-diversity and differences at the phylum and genus levels were determined, while functional pathways of classified bacteria were predicted. RESULTS: ATB influenced fecal bacterial composition and hence bacterial functional pathways before induction of colitis. After induction of colitis, ATB increased onset of clinical disease, histologic score, and colonic IL-6. In addition, ATB decreased fecal microbial richness, changed fecal and colon microbial composition, which was accompanied by a modification of microbial functional pathways. Also, several taxa were associated with ATB at lower taxonomical levels. CONCLUSIONS: The results support the hypothesis that antepartum antibiotics modulate offspring intestinal bacterial colonization and increase susceptibility to develop colonic inflammation in a murine model of colitis, and may guide future interventions to restore physiologic intestinal colonization in offspring born by antibiotic-exposed mothers.

Immunomodulatory effect of ghrelin in the intestinal mucosa.

The gastrointestinal tract is the largest endocrine organ in the body and it produces a wide array of hormones and neuropeptides. Ghrelin, a 28-amino acid hormone produced mainly by the X/A-like endocrine cells in the gastric mucosa, has widespread tissue distribution and diverse physiological functions such as hormonal, orexigenic, metabolic, cardiovascular, neurological and immunological activities. Recent research has implicated ghrelin in gastrointestinal pathological conditions and immune system regulation, but its contribution is controversial. Although ghrelin levels are elevated in clinical active inflammatory bowel diseases, confirmation of its exact role using experimental models remains unclear. This review discusses the conflicting effects of ghrelin on intestinal inflammation, through the different possible immune and intracellular mechanisms and highlights new findings.

Autophagy Is Required for Neutrophil-Mediated Inflammation.

Autophagy, an intracellular degradation and energy recycling mechanism, is emerging as an important regulator of immune responses. However, the role of autophagy in regulating neutrophil functions is not known. We investigated neutrophil biology using myeloid-specific autophagy-deficient mice and found that autophagy deficiency reduced neutrophil degranulation in vitro and in vivo. Mice with autophagy deficiency showed reduced severity of several neutrophil-mediated inflammatory and autoimmune disease models, including PMA-induced ear inflammation, LPS-induced breakdown of blood-brain barrier, and experimental autoimmune encephalomyelitis. NADPH oxidase-mediated reactive oxygen species generation was also reduced in autophagy-deficient neutrophils, and inhibition of NADPH oxidase reduced neutrophil degranulation, suggesting NADPH oxidase to be a player at the intersection of autophagy and degranulation. Overall, this study establishes autophagy as an important regulator of neutrophil functions and neutrophil-mediated inflammation in vivo.