Therapeutic Application of an Extract of Helicobacter pylori Ameliorates the Development of Allergic Airway Disease.

Epidemiological and experimental studies have shown that exposure to the gastric bacterium Helicobacter pylori, especially in early life, prevents the development of asthma. Recent mouse studies have shown that this protective effect does not require live bacteria and that treatment with an extract of H. pylori in neonates prevents the development of airway inflammation and goblet cell metaplasia. In the current study, the effect of administration of an extract of H. pylori was assessed in a therapeutic study design with application of the extract just prior to allergen challenge. C57BL/6 mice were sensitized and challenged with OVA or house dust mite. Treatment with H. pylori extract just prior to the challenge significantly reduced airway inflammation, as assessed in bronchoalveolar lavage fluid and lung tissue, and reduced airway remodeling, as assessed by goblet cell quantification. These effects were apparent in the OVA model and in the house dust mite model. Injection of H. pylori extract reduced the processing of allergen by dendritic cells in the lungs and mediastinal lymph node. Bone marrow-derived dendritic cells exposed to H. pylori extract were affected with regard to their ability to process Ag. These data show that application of H. pylori extract after sensitization effectively inhibits allergic airway disease.

The effect of tiotropium in combination with olodaterol on house dust mite-induced allergic airway disease.

One of the major goals of asthma therapy is to maintain asthma control and prevent acute exacerbations. Long-acting bronchodilators are regularly used for the treatment of asthma patients and in clinical studies the anti-cholinergic tiotropium has recently been shown to reduce exacerbations in patients with asthma. So far it is unclear how tiotropium exerts this effect. For this purpose, we designed an allergen-driven rechallenge model of allergic airway inflammation in mice, to assess the effectiveness of tiotropium and the long-acting ß-2 adrenoceptor agonist olodaterol on allergen-induced exacerbations of airway disease. Female C57BL/6J mice were sensitized intranasally (i.n.) with 1 µg of house dust mite (HDM) extract followed by a challenge regime (5 consecutive days 10 µg HDM extract i.n.) after one week. Mice were exposed to a secondary challenge five weeks after sensitization and were treated i.n. with different concentrations of tiotropium or olodaterol (1, 10 and 100 µg/kg) or a combination thereof (10 µg/kg each) prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were collected for flow cytometry and histologic analysis, respectively. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration and goblet cell metaplasia. Treatment with tiotropium, but not with olodaterol reduced tissue inflammation and goblet cell metaplasia in a dose-dependent manner. The combination of tiotropium and olodaterol was more effective in significantly reducing tissue inflammation compared to tiotropium treatment alone, and also led to a decrease in BAL cell counts. These data suggest that in a model of relapsing allergic airway disease tiotropium directly prevents exacerbations by reducing inflammation and mucus production in the airways. In addition, the combination of tiotropium and olodaterol exerts synergistic effects.

A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

There is still uncertainty whether extremely low frequency electromagnetic fields (ELF-EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF-EMF for 1, 4, and 24 h/day in a short-term (1 week) and long-term (15 weeks) set-up to investigate whole body effects on the level of stress regulation and immune response. ELF-EMF signal contained multiple frequencies (20-5000 Hz) and a magnetic flux density of 10 µT. After exposure, blood was analyzed for leukocyte numbers (short-term and long-term) and adrenocorticotropic hormone concentration (short-term only). Furthermore, in the short-term experiment, stress-related parameters, corticotropin-releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene-expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short-term but not long-term experiment, leukocyte counts were significantly higher in the 24 h-exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short-term ELF-EMF exposure may affect hypothalamic-pituitary-adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433-443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines.

The effects of low frequency electromagnetic fields (LF EMF) on human health are the subject of on-going research and serious public concern. These fields potentially elicit small effects that have been proposed to have consequences, either positive or negative, for biological systems. To reveal potentially weak but biologically relevant effects, we chose to extensively examine exposure of immune cells to two different signals, namely a complex multiple waveform field, and a 50 Hz sine wave. These immune cells are highly responsive and, in vivo, modulation of cytokine expression responses can result in systemic health effects. Using time course experiments, we determined kinetics of cytokine and other inflammation-related genes in a human monocytic leukemia cell line, THP-1, and primary monocytes and macrophages. Moreover, cytokine protein levels in THP-1 monocytes were determined. Exposure to either of the two signals did not result in a significant effect on gene and protein expression in the studied immune cells. Also, additional experiments using non-immune cells showed no effects of the signals on cytokine gene expression. We therefore conclude that these LF EMF exposure conditions are not expected to significantly modulate innate immune signaling.

Differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia.

OBJECTIVES: Endotoxin (lipopolysaccharide) tolerance is characterized by a transient refractory state to a subsequent lipopolysaccharide challenge. Following human endotoxemia, ex vivo tolerance of circulating leukocytes to lipopolysaccharide resolves within 24 hrs. However, the duration of in vivo tolerance, assumed to be primarily mediated by tissue-resident macrophages, is unknown. DESIGN, SETTING, SUBJECTS, AND INTERVENTIONS: Clinical experimental study in 16 healthy male volunteers at an intensive care research unit. To compare ex vivo and in vivo tolerance kinetics, whole blood from healthy volunteers was stimulated with lipopolysaccharide before, 4 hrs after, and 1, 2, 3, and 4 wks following in vivo endotoxin (2 ng/kg; lipopolysaccharide derived from Escherichia coli O:113) administration. Furthermore, we compared the inflammatory response during two subsequent endotoxemia experiments in healthy volunteers with an interval of 2 wks. The cytokines tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were measured. MEASUREMENTS AND MAIN RESULTS: Four hours after in vivo lipopolysaccharide administration, production of tumor necrosis factor-α, interleukin-6, and interleukin-10, but not interleukin-1 receptor antagonist in ex vivo lipopolysaccharide-stimulated whole blood was diminished. Ex vivo lipopolysaccharide tolerance completely resolved within 1 week. In contrast, in vivo lipopolysaccharide tolerance was still apparent after 2 wks. Compared to the first lipopolysaccharide administration, plasma peak levels of tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were attenuated by 46%, 36%, 45%, 10%, and 14%, respectively (all p < .05). CONCLUSIONS: While ex vivo lipopolysaccharide tolerance quickly resolves, in vivo lipopolysaccharide tolerance persists for at least 2 wks. These findings strengthen the notion that the in vivo response to lipopolysaccharide is mediated by tissue-resident macrophages and that ex vivo stimulation does not accurately reflect the in vivo innate immune response. Intervention studies utilizing the human endotoxemia model should be performed using parallel groups rather than a crossover design.

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells.

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 µT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 µT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1ß, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.

Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells.

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is a low-grade opportunistic pathogen which is considered to be associated with colon cancer. It is thought that colon polyps or tumors are the main portal of entry for this bacterium and that heparan sulfate proteoglycans (HSPGs) at the colon tumor cell surface are involved in bacterial adherence during the first stages of infection. In this study, we have shown that the histone-like protein A (HlpA) of S. gallolyticus is a genuine anchorless bacterial surface protein that binds to lipoteichoic acid (LTA) of the gram-positive cell wall in a growth phase-dependent manner. In addition, HlpA was shown to be one of the major heparin-binding proteins of S. gallolyticus able to bind to the HSPG-expressing colon tumor cell lines HCT116 and HT-29. Strikingly, although wild-type levels of HlpA appeared to contribute to adherence, coating of additional HlpA at the bacterial surface resulted in reduced binding to colon tumor cells. This may be explained by the fact that heparan sulfate and LTA compete for the same binding site in HlpA. Altogether, this study implies that HlpA serves as a fine-tuning factor for bacterial adherence.

Spleen-derived IFN-γ induces generation of PD-L1+-suppressive neutrophils during endotoxemia.

The immune inhibitory checkpoint molecule programmed death ligand (PD-L)-1 is increasingly recognized as an important player in the immune suppression observed in patients with sepsis, but its role has mainly been studied in monocytes. In an earlier study, we demonstrated that experimental human endotoxemia results in mobilization of a subset of PD-L1-expressing neutrophils displaying an IFN-γ-induced transcriptome profile. Herein, we identify the source of IFN-γ production during murine endotoxemia and its role in the generation of PD-L1+-suppressive neutrophils. We demonstrate that, similar to what we found in humans, murine endotoxemia results in the influx of a subset of PD-L1+ neutrophils in the circulation, and incubation of mouse neutrophils with recombinant IFN-γ profoundly increases PD-L1 expression. Furthermore, administration of anti-IFN-γ abrogated the generation of PD-L1+ neutrophils in endotoxemic mice. The critical involvement of the spleen is illustrated by the fact that splenectomy nullified circulating IFN-γ levels and substantially reduced the abundance of PD-L1+ neutrophils, whereas cotreatment with recombinant IFN-γ resulted in complete restoration of generation of PD-L1+ neutrophils in splenectomized mice. Finally, the functional importance of spleen-derived PD-L1+ neutrophils is exemplified by the finding that the profound decrease in T-lymphocyte proliferation observed in cells from endotoxemic mice was attenuated in cells from splenectomized animals. We demonstrated that spleen-derived IFN-γ induces generation of PD-L1+-suppressive neutrophils, implying that the spleen is critically involved in immune suppression during inflammatory diseases such as sepsis. Furthermore, our data suggest that IFN-γ plays a dual role by enhancing innate immunity and at the same time suppressing adaptive immune responses.

Acute and chronic effects of treatment with mesenchymal stromal cells on LPS-induced pulmonary inflammation, emphysema and atherosclerosis development.

BACKGROUND: COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice. METHODS: Hyperlipidemic E3L mice were intranasally instilled with 10 µg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study. RESULTS: In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment. CONCLUSION: These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study.

Microorganism-induced suppression of allergic airway disease: novel therapies on the horizon?

Allergic airway disease is a major global health burden, and novel treatment options are urgently needed. Numerous epidemiological and experimental studies suggest that certain helminths and bacteria protect against respiratory allergies. These microorganisms are strong regulators of the immune system, and various potential regulatory mechanisms by which they protect against allergic airway inflammation have been proposed. Whereas early studies addressed the beneficial effect of natural infections, the focus now shifts toward identifying the dominant protective molecules and exploring their efficacy in models of allergic airway disease. In this article, we will review the evidence for microbe-mediated protection from allergic airway disease, the potential modes of action involved and discuss advances as well as limitations in the translation of this knowledge into novel treatment strategies against allergic airway disease.

Bacterial lipopolysaccharide inhibits influenza virus infection of human macrophages and the consequent induction of CD8+ T cell immunity.

It is well established that infection with influenza A virus (IAV) facilitates secondary bacterial disease. However, there is a growing body of evidence that the microbial context in which IAV infection occurs can affect both innate and adaptive responses to the virus. To date, these studies have been restricted to murine models of disease and the relevance of these findings in primary human cells remains to be elucidated. Here, we show that pre-stimulation of primary human monocyte-derived macrophages (MDMs) with the bacterial ligand lipopolysaccharide (LPS) reduces the ability of IAV to infect these cells. The inhibition of IAV infection was associated with a reduced transcription of viral RNA and the ability of LPS to induce an anti-viral/type I interferon response in human MDMs. We demonstrated that this reduced rate of viral infection is associated with a reduced ability to present a model antigen to autologous CD8+ T cells. Taken together, these data provide the first evidence that exposure to bacterial ligands like LPS can play an important role in modulating the immune response of primary human immune cells towards IAV infection, which may then have important consequences for the development of the host's adaptive immune response.

IFN-γ-stimulated neutrophils suppress lymphocyte proliferation through expression of PD-L1.

During systemic inflammation different neutrophil subsets are mobilized to the peripheral blood. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16(bright)/CD62L(bright)), based on either an immature CD16(dim)/CD62L(bright) or a CD16(bright)/CD62L(dim) phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but how neutrophils become suppressive is unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes in mRNA expression that are relevant for their functions. Neutrophil subsets were isolated by fluorescence-activated cell sorting from blood of healthy volunteers that were administered a single dose of lipopolysaccharide (2 ng/kg i.v.) and the transcriptome was determined by microarray analysis. Interestingly, the CD16(bright)/CD62L(dim) suppressive neutrophils showed an interferon-induced transcriptome profile. More importantly, IFN-γ, but not IFN-α or IFN-ß stimulated neutrophils, acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that IFN-γ-induced expression of PD-L1 on neutrophils enables suppression of lymphocyte proliferation. Specific stimulation of neutrophils present at the inflammatory sites might therefore have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease.

Modulation of granulocyte kinetics by GM-CSF/IFN-γ in a human LPS rechallenge model.

Inflammation in response to infection or trauma can lead to CARS, which is characterized by leukocyte dysfunction. In this study, we used a human model system for CARS to study the effect of GM-CSF and IFN-γ treatment on this immunoparalyzed state. Healthy human volunteers were treated with GM-CSF (4 µg/kg), IFN-γ (100 µg), or placebo in between two challenges with Escherichia coli LPS/endotoxin (2 ng/kg). Serial leukocyte blood counts were measured. Neutrophil subsets were discriminated using CD16 and CD62L expression. LPS rechallenge resulted in increased mobilization of mature neutrophils, whereas banded neutrophils decreased. GM-CSF and IFN-γ treatment did not restore these changes. GM-CSF treatment did, however, increase the number of CD16(bright)/CD62L(dim) neutrophils that were previously shown be able to suppress T cell proliferation. IFN-γ treatment decreased neutrophilia seen after LPS rechallenge. Our study shows that LPS rechallenge was associated with changes in the distribution of neutrophil subsets, whereas no additional changes in kinetics of other granulocyte populations were observed. GM-CSF and IFN-γ treatment induced a shift in granulocyte composition toward an anti-inflammatory direction by increasing CD16(bright)/CD62L(dim) cells or decreasing neutrophil counts, respectively.

Transcriptome kinetics of circulating neutrophils during human experimental endotoxemia.

Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1ß. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.