Gut Microbiome and Transcriptomic Changes in Cigarette Smoke-Exposed Mice Compared to COPD and CD Patient Datasets.

Chronic obstructive pulmonary disease (COPD) patients and smokers have a higher incidence of intestinal disorders. The aim of this study was to gain insight into the transcriptomic changes in the lungs and intestines, and the fecal microbial composition after cigarette smoke exposure. Mice were exposed to cigarette smoke and their lung and ileum tissues were analyzed by RNA sequencing. The top 15 differentially expressed genes were investigated in publicly available gene expression datasets of COPD and Crohn's disease (CD) patients. The murine microbiota composition was determined by 16S rRNA sequencing. Increased expression of MMP12, GPNMB, CTSK, CD68, SPP1, CCL22, and ITGAX was found in the lungs of cigarette smoke-exposed mice and COPD patients. Changes in the intestinal expression of CD79B, PAX5, and FCRLA were observed in the ileum of cigarette smoke-exposed mice and CD patients. Furthermore, inflammatory cytokine profiles and adhesion molecules in both the lungs and intestines of cigarette smoke-exposed mice were profoundly changed. An altered intestinal microbiota composition and a reduction in bacterial diversity was observed in cigarette smoke-exposed mice. Altered gene expression in the murine lung was detected after cigarette smoke exposure, which might simulate COPD-like alterations. The transcriptomic changes in the intestine of cigarette smoke-exposed mice had some similarities with those of CD patients and were associated with changes in the intestinal microbiome. Future research could benefit from investigating the specific mechanisms underlying the observed gene expression changes due to cigarette smoke exposure, focusing on identifying potential therapeutic targets for COPD and CD.

Protective Effects of Alginate and Chitosan Oligosaccharides against Clostridioides difficile Bacteria and Toxin.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

Galacto-Oligosaccharides as an Anti-Infective and Anti-Microbial Agent for Macrolide-Resistant and -Sensitive Mycoplasma pneumoniae.

The worldwide increase in the incidence of antibiotic resistance of the atypical bacterium Mycoplasma pneumoniae (MP) challenges the treatment of MP infections, especially in children. Therefore, alternative strategies for the treatment of MP infections are warranted. Galacto- and fructo-oligosaccharides (GOS and FOS) are a specific group of complex carbohydrates that were recently shown to possess direct anti-pathogenic properties. In this study, we assessed whether GOS and FOS exert anti-microbial and anti-infective effects against MP and, especially, macrolide-resistant MP (MRMP) in vitro. The MIC values of GOS for MP and MRMP were 4%. In contrast, the MIC values of FOS for both MP and MRMP were 16%. A time-kill kinetic assay showed that FOS possess bacteriostatic properties, while for GOS, a bactericidal effect against MP and MRMP was observed after 24 h at a concentration of 4x MIC. In co-cultures with human alveolar A549 epithelial cells, GOS killed adherent MP and MRMP and also concentration-dependently inhibited their adherence to A549 cells. Further, GOS suppressed (MR)MP-induced IL-6 and IL-8 in A549 cells. None of the aforementioned parameters were affected when FOS were added to these co-cultures. In conclusion, the anti-infective and anti-microbial properties of GOS could provide an alternative treatment against MRMP and MP infections.

The Bidirectional Gut-Lung Axis in Chronic Obstructive Pulmonary Disease.

Epidemiological studies indicate that chronic obstructive pulmonary disease (COPD) is associated with the incidence of changes in intestinal health. Cigarette smoking, as one of the major causes of COPD, can have an impact on the gastrointestinal system and promotes intestinal diseases. This points to the existence of gut-lung interactions, but an overview of the underlying mechanisms of the bidirectional connection between the lungs and the gut in COPD is lacking. The interaction between the lungs and the gut can occur through circulating inflammatory cells and mediators. Moreover, gut microbiota dysbiosis, observed in both COPD and intestinal disorders, can lead to a disturbed mucosal environment, including the intestinal barrier and immune system, and hence may negatively affect both the gut and the lungs. Furthermore, systemic hypoxia and oxidative stress that occur in COPD may also be involved in intestinal dysfunction and play a role in the gut-lung axis. In this review, we summarize data from clinical research, animal models, and in vitro studies that may explain the possible mechanisms of gut-lung interactions associated with COPD. Interesting observations on the possibility of promising future add-on therapies for intestinal dysfunction in patients with COPD are highlighted.

Differential Effects of Oligosaccharides, Antioxidants, Amino Acids and PUFAs on Heat/Hypoxia-Induced Epithelial Injury in a Caco-2/HT-29 Co-Culture Model.

(1) Exposure of intestinal epithelial cells to heat and hypoxia causes a (heat) stress response, resulting in the breakdown of epithelial integrity. There are indications that several categories of nutritional components have beneficial effects on maintaining the intestinal epithelial integrity under stress conditions. This study evaluated the effect of nine nutritional components, including non-digestible oligosaccharides (galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), chitosan oligosaccharides (COS)), antioxidants (α-lipoic acid (ALA), resveratrol (RES)), amino acids (l-glutamine (Glu), l-arginine (Arg)) and polyunsaturated fatty acids (PUFAs) (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)), on heat/hypoxia-induced epithelial injury. (2) Two human colonic cell lines, Caco-2 and HT-29, were co-cultured and pre-treated with the nutritional components for 48 h. After pre-treatment, the cells were exposed to heat/hypoxia (42 °C, 5% O2) for 2 h. Epithelial integrity was evaluated by measuring trans-epithelial electrical resistance (TEER), paracellular Lucifer Yellow (LY) permeability, and tight junction (TJ) protein expression. Heat stress and oxidative stress levels were evaluated by determining heat-shock protein-70 (HSP-70) expression and the concentration of the lipid peroxidation product malondialdehyde (MDA). (3) GOS, FOS, COS, ALA, RES, Arg, and EPA presented protective effects on epithelial damage in heat/hypoxia-exposed Caco-2/HT-29 cells by preventing the decrease in TEER, the increase in LY permeability, and/or decrease in TJ proteins zonula occludens-1 (ZO-1) and claudin-3 expression. COS, RES, and EPA demonstrated anti-oxidative stress effects by suppressing the heat/hypoxia-induced MDA production, while Arg further elevated the heat/hypoxia-induced increase in HSP-70 expression. (4) This study indicates that various nutritional components have the potential to counteract heat/hypoxia-induced intestinal injury and might be interesting candidates for future in vivo studies and clinical trials in gastrointestinal disorders related to heat stress and hypoxia.

Effects of a nutritional intervention on impaired behavior and cognitive function in an emphysematous murine model of COPD with endotoxin-induced lung inflammation.

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.

Non-Digestible Oligosaccharides: A Novel Treatment for Respiratory Infections?

Emerging antimicrobial resistance in respiratory infections requires novel intervention strategies. Non-digestible oligosaccharides (NDOs) are a diverse group of carbohydrates with broad protective effects. In addition to promoting the colonization of beneficial gut microbiota and maintaining the intestinal homeostasis, NDOs act as decoy receptors, effectively blocking the attachment of pathogens on host cells. NDOs also function as a bacteriostatic agent, inhibiting the growth of specific pathogenic bacteria. Based on this fact, NDOs potentiate the actions of antimicrobial drugs. Therefore, there is an increasing interest in characterizing the anti-infective properties of NDOs. This focused review provides insights into the mechanisms by which representative NDOs may suppress respiratory infections by targeting pathogens and host cells. We summarized the most interesting mechanisms of NDOs, including maintenance of gut microbiota homeostasis, interference with TLR-mediated signaling, anti-oxidative effects and bacterial toxin neutralization, bacteriostatic and bactericidal effects, and anti-adhesion or anti-invasive properties. A detailed understanding of anti-infective mechanisms of NDOs against respiratory pathogens may contribute to the development of add-on therapy or alternatives to antimicrobials.

Direct Action of Non-Digestible Oligosaccharides against a Leaky Gut.

The epithelial monolayer is the primary determinant of mucosal barrier function, and tight junction (TJ) complexes seal the paracellular space between the adjacent epithelial cells and represent the main "gate-keepers" of the paracellular route. Impaired TJ functionality results in increased permeation of the "pro-inflammatory" luminal contents to the circulation that induces local and systemic inflammatory and immune responses, ultimately triggering and/or perpetuating (chronic) systemic inflammatory disorders. Increased gut leakiness is associated with intestinal and systemic disease states such as inflammatory bowel disease and neurodegenerative diseases such as Parkinson's disease. Modulation of TJ dynamics is an appealing strategy aiming at inflammatory conditions associated with compromised intestinal epithelial function. Recently there has been a growing interest in nutraceuticals, particularly in non-digestible oligosaccharides (NDOs). NDOs confer innumerable health benefits via microbiome-shaping and gut microbiota-related immune responses, including enhancement of epithelial barrier integrity. Emerging evidence supports that NDOs also exert health-beneficial effects on microbiota independently via direct interactions with intestinal epithelial and immune cells. Among these valuable features, NDOs promote barrier function by directly regulating TJs via AMPK-, PKC-, MAPK-, and TLR-associated pathways. This review provides a comprehensive overview of the epithelial barrier-protective effects of different NDOs with a special focus on their microbiota-independent modulation of TJs.

Effects of Cigarette Smoke on Adipose and Skeletal Muscle Tissue: In Vivo and In Vitro Studies.

Chronic obstructive pulmonary disease (COPD), often caused by smoking, is a chronic lung disease with systemic manifestations including metabolic comorbidities. This study investigates adaptive and pathological alterations in adipose and skeletal muscle tissue following cigarette smoke exposure using in vivo and in vitro models. Mice were exposed to cigarette smoke or air for 72 days and the pre-adipose cell line 3T3-L1 was utilized as an in vitro model. Cigarette smoke exposure decreased body weight, and the proportional loss in fat mass was more pronounced than the lean mass loss. Cigarette smoke exposure reduced adipocyte size and increased adipocyte numbers. Adipose macrophage numbers and associated cytokine levels, including interleukin-1ß, interleukine-6 and tumor necrosis factor-α were elevated in smoke-exposed mice. Muscle strength and protein synthesis signaling were decreased after smoke exposure; however, muscle mass was not changed. In vitro studies demonstrated that lipolysis and fatty acid oxidation were upregulated in cigarette smoke-exposed pre-adipocytes. In conclusion, cigarette smoke exposure induces a loss of whole-body fat mass and adipose atrophy, which is likely due to enhanced lipolysis.

Galacto-oligosaccharides alleviate lung inflammation by inhibiting NLRP3 inflammasome activation in vivo and in vitro.

INTRODUCTION: The lack of effective anti-inflammatory therapies for pneumonia represents a challenge for identifying new alternatives. Non-digestible galacto-oligosaccharides (GOS) are attractive candidates due to their anti-inflammatory and immunomodulatory effects both locally and systemically. OBJECTIVES: The anti-inflammatory properties of GOS were investigated in calves with lung infections and in calf primary bronchial epithelial cells (PBECs) and human lung epithelial cells (A549). To delineate the mechanism, the potential capacity of GOS to inhibit the NLR family pyrin domain containing 3 (NLRP3) inflammasome has been investigated. METHODS: GOS were administrated orally to calves with naturally occurring lung infections during early life or used as pretreatments in cell cultures exposed to M. haemolytica, lipopolysaccharides (LPS), leukotoxin or ATP. The cell composition, cytokine/chemokine concentrations, and M. haemolytica-LPS lgG levels in broncho-alveolar lavage fluid (BALF) and blood were investigated, while the M. haemolytica positivity in BALF and bronchial mucosa was detected in vivo. Key markers of NLRP3 inflammasome activation were measured in vivo and in vitro. RESULTS: GOS reduced M. haemolytica positivity and M. haemolytica-LPS lgG levels in calves with lung infections. Regulation of immune function and suppression of inflammatory response by GOS is related to the inhibition of NLRP3 inflammasome as observed in bronchial mucosal tissue of infected calves. The M. haemolytica-induced IL-1ß production in PBECs was lowered by GOS, which was associated with NLRP3 inflammasome inhibition caused by the decreased reactive oxygen species and ATP production. GOS inhibited leukotoxin-induced ATP production in PBECs. The LPS- and ATP-induced NLRP3 inflammasome activation in PBECs and A549 cells was suppressed by GOS. CONCLUSION: GOS exert anti-inflammatory properties by inhibiting the NLRP3 inflammasome activation in vitro and in vivo, suggesting a potential role for GOS in the prevention of lung infections.

Deoxynivalenol exposure during pregnancy has adverse effects on placental structure and immunity in mice model.

Deoxynivalenol (DON), a highly prevalent food contaminant, is known to induce reproductive and immunotoxicity in humans upon exposure. The present study focused on the consequences of exposure to DON during pregnancy for placental barrier and immune function, as well as fetal survival. Female mice received diets contaminated with DON (6.25 and 12.5 mg/kg of diet), starting immediately after mating until the end of the experiment. On day 17 of pregnancy the animals were killed, and maternal and fetal samples were collected for further analysis. Feeding on DON-contaminated diets decreased fetal survival, and DON was detected at significant levels in the fetus. Placentae from DON-exposed mice revealed a reduction in expression of junctional proteins, ZO-1, E-cadherin and claudins, upregulation of AHR mRNA expressions, and increase in IFN-ꝩ, IL-6 and IL-4 production. In conclusion, results of this study demonstrate harmful effects of DON on the course of pregnancy and fetal survival, which might be due to immunological changes in maternal immune organs and placenta. Altogether, these data underline the importance of the quality of maternal diet during pregnancy as they clearly demonstrate the potential harmful effects of a commonly present food-contaminant.

Changes in intestinal homeostasis and immunity in a cigarette smoke- and LPS-induced murine model for COPD: the lung-gut axis.

Chronic obstructive pulmonary disease (COPD) is often associated with intestinal comorbidities. In this study, changes in intestinal homeostasis and immunity in a cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced COPD model were investigated. Mice were exposed to cigarette smoke or air for 72 days, except days 42, 52, and 62 on which the mice were treated with saline or LPS via intratracheal instillation. Cigarette smoke exposure increased the airway inflammatory cell numbers, mucus production, and different inflammatory mediators, including C-reactive protein (CRP) and keratinocyte-derived chemokine (KC), in bronchoalveolar lavage (BAL) fluid and serum. LPS did not further impact airway inflammatory cell numbers or mucus production but decreased inflammatory mediator levels in BAL fluid. T helper (Th) 1 cells were enhanced in the spleen after cigarette smoke exposure; however, in combination with LPS, cigarette exposure caused an increase in Th1 and Th2 cells. Histomorphological changes were observed in the proximal small intestine after cigarette smoke exposure, and addition of LPS had no effect. Cigarette smoke activated the intestinal immune network for IgA production in the distal small intestine that was associated with increased fecal sIgA levels and enlargement of Peyer's patches. Cigarette smoke plus LPS decreased fecal sIgA levels and the size of Peyer's patches. In conclusion, cigarette smoke with or without LPS affects intestinal health as observed by changes in intestinal histomorphology and immune network for IgA production. Elevated systemic mediators might play a role in the lung-gut cross talk. These findings contribute to a better understanding of intestinal disorders related to COPD.

Increased exploration and hyperlocomotion in a cigarette smoke and LPS-induced murine model of COPD: linking pulmonary and systemic inflammation with the brain.

Brain-related comorbidities are frequently observed in chronic obstructive pulmonary disease (COPD) and are related to increased disease progression and mortality. To date, it is unclear which mechanisms are involved in the development of brain-related problems in COPD. In this study, a cigarette smoke and lipopolysaccharide (LPS) exposure murine model was used to induce COPD-like features and assess the impact on brain and behavior. Mice were daily exposed to cigarette smoke for 72 days, except for days 42, 52, and 62, on which mice were intratracheally exposed to the bacterial trigger LPS. Emphysema and pulmonary inflammation as well as behavior and brain pathology were assessed. Cigarette smoke-exposed mice showed increased alveolar enlargement and numbers of macrophages and neutrophils in bronchoalveolar lavage. Cigarette smoke exposure resulted in lower body weight, which was accompanied by lower serum leptin levels, more time spent in the inner zone of the open field, and decreased claudin-5 and occludin protein expression levels in brain microvessels. Combined cigarette smoke and LPS exposure resulted in increased locomotion and elevated microglial activation in the hippocampus of the brain. These novel findings show that systemic inflammation observed after combined cigarette smoke and LPS exposure in this COPD model is associated with increased exploratory behavior. Findings suggest that neuroinflammation is present in the brain area involved in cognitive functioning and that blood-brain barrier integrity is compromised. These findings can contribute to our knowledge about possible processes involved in brain-related comorbidities in COPD, which is valuable for optimizing and developing therapy strategies.

Repeated exposure of bronchial epithelial cells to particular matter increases allergen-induced cytokine release and permeability.

Long term particulate matter (PM) exposure has been associated with an increased incidence of respiratory diseases. Here, an in vitro model was developed to study how long term diesel exhaust particle (DEP) exposure might predispose to the development of allergic reactions. Airway epithelial (16HBE) cells were exposed to low concentrations of diesel exhaust particle (DEP) for 4 days after which they were challenged with house dust mite (HDM) extract (24 h). Compared to acute exposure (24 h), 4 days DEP exposure to 16HBE cells further reduced the transepithelial electrical resistance (TEER) and increased CXCL-8 release. DEP pre-exposure aggravated HDM-induced loss of TEER, increased tracer flux across the barrier and reduced CLDN-3 expression in these 16HBE cells. HDM-induced cytokine (IL-6, CCL-22, IL-10 and CXCL-8) release was significantly increased after DEP pre-exposure. In the current study an in vitro model with long term PM exposure was presented, which might be helpful for further understanding the interplay between long term PM exposure and allergic responses.

Galacto-oligosaccharides as an anti-bacterial and anti-invasive agent in lung infections.

Emerging antimicrobial resistance in infections asks for novel intervention strategies. Galacto-oligosaccharides (GOS) might be attractive alternatives to antibiotics due to their anti-inflammatory and anti-adhesive properties. Mannheimia haemolytica is one of the major Pasteurellaceae associated with bovine lung infections. Using M. haemolytica, we demonstrated that GOS have the capacity to reduce bacterial viability and can be used as adjuvant to improve antibiotic efficacy. Using M. haemolytica-treated primary bronchial epithelial cells (PBECs) of calves, we identified the anti-adhesive and anti-invasive activities of GOS. The observed inhibition of cytokine/chemokine release and the prevention of airway epithelial barrier dysfunction in M. haemolytica-treated PBECs by GOS might be related to the downregulation of "toll-like receptor 4/nuclear factor-κB" pathway and the anti-invasive and anti-adhesive properties of GOS. Particularly, GOS lowered lipopolysaccharides- but not flagellin-induced cytokine/chemokine release in calf and human airway epithelial cells. Finally, we performed in vivo experiments in calves and demonstrated for the first time that intranasal application of GOS can relieve lung infections/inflammation and lower M. haemolytica positivity in the lungs without affecting clinical performance. These findings not only shed light on the anti-inflammatory mechanisms of GOS during lung infections, but GOS might also be a promising anti-bacterial agent for preventing (lung) infections.

Probiotics, prebiotics, and synbiotics to prevent or combat air pollution consequences: The gut-lung axis.

Air pollution exposure is a public health emergency, which attributes globally to an estimated seven million deaths on a yearly basis We are all exposed to air pollutants, varying from ambient air pollution hanging over cities to dust inside the home. It is a mixture of airborne particulate matter and gases that can be subdivided into three categories based on particle diameter. The smallest category called PM0.1 is the most abundant. A fraction of the particles included in this category might enter the blood stream spreading to other parts of the body. As air pollutants can enter the body via the lungs and gut, growing evidence links its exposure to gastrointestinal and respiratory impairments and diseases, like asthma, rhinitis, respiratory tract infections, Crohn's disease, ulcerative colitis, and abdominal pain. It has become evident that there exists a crosstalk between the respiratory and gastrointestinal tracts, commonly referred to as the gut-lung axis. Via microbial secretions, metabolites, immune mediators and lipid profiles, these two separate organ systems can influence each other. Well-known immunomodulators and gut health stimulators are probiotics, prebiotics, together called synbiotics. They might combat air pollution-induced systemic inflammation and oxidative stress by optimizing the microbiota composition and microbial metabolites, thereby stimulating anti-inflammatory pathways and strengthening mucosal and epithelial barriers. Although clinical studies investigating the role of probiotics, prebiotics, and synbiotics in an air pollution setting are lacking, these interventions show promising health promoting effects by affecting the gastrointestinal- and respiratory tract. This review summarizes the current data on how air pollution can affect the gut-lung axis and might impact gut and lung health. It will further elaborate on the potential role of probiotics, prebiotics and synbiotics on the gut-lung axis, and gut and lung health.

Intratracheal administration of solutions in mice; development and validation of an optimized method with improved efficacy, reproducibility and accuracy.

Animal models are still vital in the field of respiratory disease research. To improve the accuracy and consistency of the dose of specific compounds administered specifically in the respiratory tract, it is important to optimize and to compare the technique to currently available techniques. In this study, an optimized intubation-mediated intratracheal administration (IMIT) technique is described and compared to oropharyngeal aspiration (OA). Adult female Balb/c mice were treated with Evans Blue using IMIT or OA and sacrificed after a short recovery to observe the distribution of solutions throughout the lungs. Additionally, mice were treated with increasing doses of lipopolysaccharide (LPS) or saline to compare efficacy of both techniques. Inflammatory cell numbers in bronchoalveolar lavage were quantified 24 h post-administration. Evans Blue staining revealed a more homogeneous distribution and less variability among animals treated using IMIT as compared to OA. Higher inflammatory cell numbers were observed in IMIT mice compared to OA mice after exposure to vehicle or the lowest LPS concentration. This study shows that the optimized IMIT is superior to OA with regards to efficacy, reproducibility and accuracy. This IMIT method can be deployed to refine 3R animal welfare aspects of the experimental design and improve the reproducibility of respiratory disease mouse models.

Epithelial integrity, junctional complexes, and biomarkers associated with intestinal functions.

An intact intestinal barrier is crucial for immune homeostasis and its impairment activates the immune system and may result in chronic inflammation. The epithelial cells of the intestinal barrier are connected by tight junctions, which form an anastomosing network sealing adjacent epithelial cells. Tight junctions are composed of transmembrane and cytoplasmic scaffolding proteins. Transmembrane tight junction proteins at the apical-lateral membrane of the cell consist of occludin, claudins, junctional adhesion molecules, and tricellulin. Cytoplasmic scaffolding proteins, including zonula occludens, cingulin and afadin, provide a direct link between transmembrane tight junction proteins and the intracellular cytoskeleton. Each individual component of the tight junction network closely interacts with each other to form an efficient intestinal barrier. This review aims to describe the molecular structure of intestinal epithelial tight junction proteins and to characterize their organization and interaction. Moreover, clinically important biomarkers associated with impairment of gastrointestinal integrity are discussed.

Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells.

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.