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.

Transcriptomic profiling of the acute mucosal response to local food injections in adults with eosinophilic esophagitis.

BACKGROUND: Exposure of the esophageal mucosa to food allergens can cause acute mucosal responses in patients with eosinophilic esophagitis (EoE), but the underlying local immune mechanisms driving these acute responses are not well understood. OBJECTIVE: We sought to gain insight into the early transcriptomic changes that occur during an acute mucosal response to food allergens in EoE. METHODS: Bulk RNA sequencing was performed on esophageal biopsy specimens from adult patients with EoE (n = 5) collected before and 20 minutes after intramucosal injection of various food extracts in the esophagus. Baseline biopsy specimens from control subjects without EoE (n = 5) were also included. RESULTS: At baseline, the transcriptome of the patients with EoE showed increased expression of genes related to an EoE signature. After local food injection, we identified 40 genes with a potential role in the early immune response to food allergens (most notably CEBPB, IL1B, TNFSF18, PHLDA2, and SLC15A3). These 40 genes were enriched in processes related to immune activation, such as the acute-phase response, cellular responses to external stimuli, and cell population proliferation. TNFSF18 (also called GITRL), a member of the TNF superfamily that is best studied for its costimulatory effect on T cells, was the most dysregulated early EoE gene, showing a 12-fold increase compared with baseline and an 18-fold increase compared with a negative visual response. Further experiments showed that the esophageal epithelium may be an important source of TNFSF18 in EoE, which was rapidly induced by costimulating esophageal epithelial cells with the EoE-relevant cytokines IL-13 and TNF-α. CONCLUSIONS: Our data provide unprecedented insight into the transcriptomic changes that mediate the acute mucosal immune response to food allergens in EoE and suggest that TNFSF18 may be an important effector molecule in this response.

The relationship between immune fitness and saliva biomarkers of systemic inflammation.

Purpose: It is vital that Immune fitness, i.e., how well the immune system functions and reacts to challenges, can be reliably be examined. The current study aimed to compare immune fitness with assessments of saliva biomarkers of systemic inflammation. Methods: N = 108 healthy young adults (18-30-year-old students of Utrecht University, the Netherlands) participated in the study. A saliva sample was collected for biomarker assessment (Interleukin (IL)-1ß, IL-6, IL-8, IL-10, immunoglobulin A (IgA), and tumor necrosis factor-alpha (TNF-α), and c-reactive protein (CRP). Additionally, a survey was completed to assess immune fitness, mood, mental resilience, and quality of life. The correlations between the biomarker assessments, immune fitness and mood were determined. Results: No significant correlations between immune fitness and biomarkers of systemic inflammation were found. Significant sex differences in correlations with immune fitness were demonstrated for loneliness (significant only in men) and fatigue (significant only in women). For both sexes, immune fitness correlated significantly with anxiety, mental resilience, and quality of life. Conclusion: No significant correlations were found between immune fitness and saliva biomarkers of systemic inflammation. Immune fitness correlated significantly with anxiety, mental resilience, and quality of life. Sex differences were demonstrated in the relation of immune fitness with loneliness and fatigue. Future research should further investigate factors that may influence the relationship between immune fitness, mood, and biomarkers of systemic inflammation, including underlying psychological mechanisms of possible sex differences.

An evening of alcohol consumption negatively impacts next-day immune fitness in both hangover-sensitive drinkers and hangover-resistant drinkers.

BACKGROUND: Survey research found poorer baseline immune fitness for self-reported hangover-sensitive drinkers compared to hangover-resistant drinkers. However, up to now a limited number of clinical studies revealed mixed results regarding the relationship between the concentrations of biomarkers of systemic inflammation in blood or saliva with hangover severity, and could not differentiate between hangover-sensitive drinkers and hangover-resistant drinkers. The aim of this study was to assess immune fitness and saliva biomarkers of systemic inflammation at multiple timepoints following an alcohol day and alcohol-free control day. METHODS: The study had a semi-naturalistic design. In the evening before the test days, participants were not supervised. They could drink ad libitum drinking on the alcohol test day and refrained from drinking alcohol on the control day. Activities and behaviors on the alcohol and control day were reported the follow morning. On both test days, from 09:30 to 15:30, hourly assessments of immune fitness (single-item scale) and overall hangover severity (single-item scale) were made and saliva samples were collected for biomarker assessments. RESULTS: N = 14 hangover-resistant drinkers and n = 15 hangover-sensitive drinkers participated in the study. The amount of alcohol consumed on the alcohol day did not significantly differ between the hangover-resistant group (mean (SD) of 13.5 (7.9) alcoholic drinks) and the hangover-sensitive group (mean (SD) of 12.4 (4.4) alcoholic drinks). All hangover-sensitive drinkers reported having a hangover following the alcohol day (overall hangover severity score 6.1 (on a 0-10 scale) at 09:30, gradually decreasing to 3.3 at 15:30), whereas the hangover-resistant drinkers reported no hangover. On the control day, immune fitness of the hangover-sensitive group was significantly poorer than the hangover-resistant group. On the alcohol day, both groups showed a significant reduction in immune fitness. The effect was evident throughout the day, but significantly more pronounced in the hangover-sensitive group than the hangover-resistant group. No significant differences between the groups were found at any time point on the two test days for saliva concentrations of Interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α. CONCLUSIONS: Whereas hangover-sensitive drinkers reported a hangover following an alcohol day and hangover-resistant drinkers did not, both groups reported significantly reduced immune fitness throughout the day. However, the reduction in immune fitness among hangover-sensitive drinkers was significantly more pronounced in comparison to the hangover-resistant group.

Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption.

Low-grade inflammation and barrier disruption are increasingly acknowledged for their association with non-communicable diseases (NCDs). Short chain fatty acids (SCFAs), especially butyrate, could be a potential treatment because of their combined anti-inflammatory and barrier- protective capacities, but more insight into their mechanism of action is needed. In the present study, non-activated, lipopolysaccharide-activated and αCD3/CD28-activated peripheral blood mononuclear cells (PBMCs) with and without intestinal epithelial cells (IEC) Caco-2 were used to study the effect of butyrate on barrier function, cytokine release and immune cell phenotype. A Caco-2 model was used to compare the capacities of butyrate, propionate and acetate and study their mechanism of action, while investigating the contribution of lipoxygenase (LOX), cyclooxygenase (COX) and histone deacetylase (HDAC) inhibition. Butyrate protected against inflammatory-induced barrier disruption while modulating inflammatory cytokine release by activated PBMCs (interleukin-1 beta↑, tumor necrosis factor alpha↓, interleukin-17a↓, interferon gamma↓, interleukin-10↓) and immune cell phenotype (regulatory T-cells↓, T helper 17 cells↓, T helper 1 cells↓) in the PBMC/Caco-2 co-culture model. Similar suppression of immune activation was shown in absence of IEC. Butyrate, propionate and acetate reduced inflammatory cytokine-induced IEC activation and, in particular, butyrate was capable of fully protecting against cytokine-induced epithelial permeability for a prolonged period. Different HDAC inhibitors could mimic this barrier-protective effect, showing HDAC might be involved in the mechanism of action of butyrate, whereas LOX and COX did not show involvement. These results show the importance of sufficient butyrate levels to maintain intestinal homeostasis.

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.

Raw milk kefir: microbiota, bioactive peptides, and immune modulation.

This study aims to characterize the microbiota and peptidomic composition of raw milk kefir, and to address the potential anti-allergic effects of raw milk kefir using validated research models for food allergy. Raw milk kefir was produced after incubation with a defined freeze-dried starter culture. Kefir was sampled during fermentation at seven time intervals. For comparison, kefir was also prepared from heat-treated milk. Peptide compositions were determined for the raw and heated milk, and kefir end products made from these milks. In a murine food allergy model, the two kefir end products were investigated for their allergy modulating effects. In both kefirs, we identified amplicon sequence variants identical to those in the starter culture, matching the bacteria Lactococcus lactis, Streptococcus thermophilus, Leuconostoc and the yeast Debaryomyces. In raw milk kefir, additional sequence variants of Lactococcus lactis and the yeasts Pichia and Galactomyces could be identified, which were absent in heated milk kefir. Analysis of peptide compositions in both kefirs indicated that the number and intensity of peptides drastically increased after fermentation. Heating of the milk negatively affected the diversity of the peptide composition in kefir. Only raw milk kefir suppressed the acute allergic skin response to the food allergen ovalbumin in sensitised mice. These effects coincided with differences in the T-cell compartment, with lower percentages of activated Th1 cells and IFNg production after treatment with kefir made from heated milk. The results of this study indicate specific properties of raw milk kefir that may contribute to its additional health benefits.

The interaction between intestinal bacterial metabolites and phosphatase and tensin homolog in autism spectrum disorder.

Intestinal bacteria-associated para-cresyl sulfate (pCS) and 4-ethylphenyl sulfate (4EPS) are elevated in autism spectrum disorder (ASD). Both metabolites can induce ASD-like behaviors in mice, but the molecular mechanisms are not known. Phosphatase and tensin homolog (PTEN) is a susceptibility gene for ASD. The present study investigated the relation between pCS and 4EPS and PTEN in ASD in a valproic acid (VPA)-induced murine ASD model and an in vitro LPS-activated microglial model. The VPA-induced intestinal inflammation and compromised permeability in the distal ileum was not associated with changes of PTEN expression and phosphorylation. In contrast, VPA reduced PTEN expression in the hippocampus of mice. In vitro results show that pCS and 4EPS reduced PTEN expression and derailed innate immune response of BV2 microglial cells. The PTEN inhibitor VO-OHpic did not affect innate immune response of microglial cells. In conclusion, PTEN does not play a role in intestinal inflammation and compromised permeability in VPA-induced murine model for ASD. Although pCS and 4EPS reduced PTEN expression in microglial cells, PTEN is not involved in the pCS and 4EPS-induced derailed innate immune response of microglial cells. Further studies are needed to investigate the possible involvement of reduced PTEN expression in the ASD brain regarding synapse function and neuronal connectivity.

Immunophenotype and function of circulating myeloid derived suppressor cells in COVID-19 patients.

The pathogenesis of coronavirus disease 2019 (COVID-19) is not fully elucidated. COVID-19 is due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes severe illness and death in some people by causing immune dysregulation and blood T cell depletion. Increased numbers of myeloid-derived suppressor cells (MDSCs) play a diverse role in the pathogenesis of many infections and cancers but their function in COVID-19 remains unclear. To evaluate the function of MDSCs in relation with the severity of COVID-19. 26 PCR-confirmed COVID-19 patients including 12 moderate and 14 severe patients along with 11 healthy age- and sex-matched controls were enrolled. 10 ml whole blood was harvested for cell isolation, immunophenotyping and stimulation. The immunophenotype of MDSCs by flow cytometry and T cells proliferation in the presence of MDSCs was evaluated. Serum TGF-ß was assessed by ELISA. High percentages of M-MDSCs in males and of P-MDSCs in female patients were found in severe and moderate affected patients. Isolated MDSCs of COVID-19 patients suppressed the proliferation and intracellular levels of IFN-γ in T cells despite significant suppression of T regulatory cells but up-regulation of precursor regulatory T cells. Serum analysis shows increased levels of TGF-ß in severe patients compared to moderate and control subjects (HC) (P = 0.003, P < 0.0001, respectively). The frequency of MDSCs in blood shows higher frequency among both moderate and severe patients and may be considered as a predictive factor for disease severity. MDSCs may suppress T cell proliferation by releasing TGF-ß.

Evaluation of Myeloid-derived Suppressor Cells in the Blood of Iranian COVID-19 Patients.

The cytokine storm and lymphopenia are reported in coronavirus disease 2019 (COVID-19). Myeloid-derived suppressive cells (MDSCs) exist in two different forms, granulocyte (G-MDSCs) and monocytic (M-MDSCs), that both suppress T-cell function. In COVID-19, the role of chemokines such as interleukin (IL)-8 in recruiting MDSCs is unclear. A recent report has correlated IL-8 and MDSCs with poor clinical outcomes in melanoma patients. In the current study, we evaluated the frequency of MDSCs and their correlation with serum IL-8 levels in severe COVID-19 patients from Iran. Thirty-seven severe patients (8 on ventilation, 29 without ventilation), thirteen moderate COVID-19 patients, and eight healthy subjects participated in this study between 10th April 2020 and 9th March 2021. Clinical and biochemical features, serum, and whole blood were obtained. CD14, CD15, CD11b, and HLA-DR expression on MDSCs was measured by flow cytometry. COVID-19 patients compared to healthy subjects had a greater frequency of M-MDSCs (12.7±13.3% vs 0.19±0.20%,), G-MDSCs (15.8±12.6% vs 0.35±0.40%,) and total-MDSCs (27.5±17.3% vs 0.55±0.41%,). M-MDSC (16.8±15.8% vs 5.4±4.8%,) and total-MDSC (33.3±18.5% vs 17.3±13.3%) frequency was higher in non- ventilated compared to moderate COVID-19 subjects. Serum IL-8 levels were higher in patients with COVID-19 than in normal healthy subjects (6.4±7.8 vs. 0.10±00 pg/mL). Ventilated patients (15.7±6.7 pg/mL), non-ventilated patients (5.7±2.7 pg/mL) and moderate patients (2.8±3.0 pg/mL) had significantly different levels of IL-8.  A negative correlation was found between the frequency of G-MDSCs and the international normalized ratio (INR) test (r=-0.39), and between the frequency of total-MDSCs and oxygen saturation (%) (r=-0.39). COVID-19 patients with severe non-ventilated disease had the highest levels of M-MDSCs. In addition to systemic MDSCs, lung, serum IL-8, and other inflammatory biomarkers should be measured.

The Autism Spectrum Disorder-Associated Bacterial Metabolite p-Cresol Derails the Neuroimmune Response of Microglial Cells Partially via Reduction of ADAM17 and ADAM10.

The bacterial metabolite 4-methylphenol (para-cresol or p-cresol) and its derivative p-cresyl sulfate (pCS) are elevated in the urine and feces of children with autism spectrum disorder (ASD). It has been shown that p-cresol administration induces social behavior deficits and repetitive behavior in mice. However, the mechanisms of p-cresol, specifically its metabolite pCS that can reach the brain, in ASD remain to be investigated. The pCS has been shown to inhibit LPS-stimulated inflammatory response. A Disintegrin And Metalloprotease 10 (ADAM10) and A Disintegrin And Metalloprotease 17 (ADAM17) are thought to regulate microglial immune response by cleaving membrane-bound proteins. In the present study, a neuroinflammation model of LPS-activated BV2 microglia has been used to unveil the potential molecular mechanism of pCS in ASD pathogenesis. In microglial cells pCS treatment decreases the expression or maturation of ADAM10 and ADAM17. In addition, pCS treatment attenuates TNF-α and IL-6 releases as well as phagocytosis activity of microglia. In in vitro ADAM10/17 inhibition experiments, either ADAM10 or ADAM17 inhibition reduces constitutive and LPS-activated release of TNF-α, TNFR-1 and IL-6R by microglial cells, while it increases constitutive and LPS-activated microglial phagocytotic activity. The in vivo results further confirm the involvement of ADAM10 and ADAM17 in ASD pathogenesis. In in utero VPA-exposed male mice, elevated concentration in serum of p-cresol-associated metabolites pCS and p-cresyl glucuronide (pCG) is associated with a VPA-induced increased ADAM10 maturation, and a decreased ADAM17 maturation that is related with attenuated levels of soluble TNF-α and TGF-ß1 in the mice brain. Overall, the present study demonstrates a partial role of ADAM10 and ADAM17 in the derailed innate immune response of microglial cells associated with pCS-induced ASD pathogenesis.

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.

Human Milk Oligosaccharide 2'-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells.

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2'-fucosyllactose (2'FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2'FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4+ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNß, IFNλ1 and CCL5 secretion. Pre-incubation with 2'FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2'FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2'FL. These results provide insights regarding the protective capacity of human milk components in early life.

Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death.

Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.

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.

Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition.

Non-communicable diseases are increasing and have an underlying low-grade inflammation in common, which may affect gut health. To maintain intestinal homeostasis, unwanted epithelial activation needs to be avoided. This study compared the efficacy of butyrate, propionate and acetate to suppress IFN-γ+/-TNF-α induced intestinal epithelial activation in association with their HDAC inhibitory capacity, while studying the canonical and non-canonical STAT1 pathway. HT-29 were activated with IFN-γ+/-TNF-α and treated with short chain fatty acids (SCFAs) or histone deacetylase (HDAC) inhibitors. CXCL10 release and protein and mRNA expression of proteins involved in the STAT1 pathway were determined. All SCFAs dose-dependently inhibited CXCL10 release of the cells after activation with IFN-γ or IFN-γ+TNF-α. Butyrate was the most effective, completely preventing CXCL10 induction. Butyrate did not affect phosphorylated STAT1, nor phosphorylated NFκB p65, but inhibited IRF9 and phosphorylated JAK2 protein expression in activated cells. Additionally, butyrate inhibited CXCL10, SOCS1, JAK2 and IRF9 mRNA in activated cells. The effect of butyrate was mimicked by class I HDAC inhibitors and a general HDAC inhibitor Trichostatin A. Butyrate is the most potent inhibitor of CXCL10 release compared to other SCFAs and acts via HDAC inhibition. This causes downregulation of CXCL10, JAK2 and IRF9 genes, resulting in a decreased IRF9 protein expression which inhibits the non-canonical pathway and CXCL10 transcription.

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.

Esterified derivatives of DHA and EPA increase bortezomib cytotoxicity in human multiple myeloma cells.

BACKGROUND & AIMS: Although the proteasome inhibitor bortezomib has greatly improved the clinical outcome of patients with multiple myeloma (MM), acquired drug resistance remains the greatest obstacle on the road of treating MM. We previously showed that omega-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) with the chemotherapeutic agent bortezomib can overcome its chemoresistance in MM cells. However, most DHA/EPA are esterified shortly after oral administration, which may affect their bioactivity. This study was to evaluate the cytotoxicity of ethyl ester-DHA/EPA in human MM cells. The mechanisms relevant for the cytotoxicity of these esterified-fatty acids were further investigated. METHODS: Human MM cell lines L363, OPM2, U266 were treated with ethyl ester-DHA/EPA with or without bortezomib. The percentage of dead cells and intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. RESULTS: Ethyl ester-DHA and -EPA were much more potent than DHA/EPA to induce cytotoxicity in MM cells, even in DHA/EPA-resistant MM cells. Pretreating MM cells with esterified-DHA/EPA before bortezomib potently increased its cytotoxicity. Additionally, intracellular ROS levels were upregulated in MM cells after treatment with ethyl ester-DHA/EPA, which reflected the enhanced oxidative stress in treated cells. CONCLUSIONS: This study provides evidence that ethyl ester-DHA/EPA in combination with bortezomib may improve the overall efficacy in MM cells, similar to DHA/EPA, relieving the concern that esterification of DHA/EPA may affect its bioactivity and further supporting the potential clinical use of fatty acids DHA/EPA for combating drug resistance during MM therapy.

Modulation of the Epithelial-Immune Cell Crosstalk and Related Galectin Secretion by DP3-5 Galacto-Oligosaccharides and ß-3'Galactosyllactose.

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized ß-3'galactosyllactose (ß-3'GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or ß-3'GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized ß-3'GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by ß-3'GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized ß-3'GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.