The surface-associated exopolysaccharide of Bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro. In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses

TLR4-induced IFN-γ production increases TLR2 sensitivity and drives Gram-negative sepsis in mice

Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4–MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-α release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-γ in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection

Electrical impedance spectroscopy detects skin barrier dysfunction in childhood atopic dermatitis

Background Skin barrier dysfunction is associated with the development of atopic dermatitis (AD), however methods to assess skin barrier function are limited. We investigated the use of electrical impedance spectroscopy (EIS) to detect skin barrier dysfunction in children with AD of the CARE (Childhood AlleRgy, nutrition, and Environment) cohort. Methods EIS measurements taken at multiple time points from 4 months to 3‐year‐old children, who developed AD (n = 66) and those who did not (n = 49) were investigated. Using only the EIS measurement and the AD status, we developed a machine learning algorithm that produces a score (EIS/AD score) which reflects the probability that a given measurement is from a child with active AD. We investigated the diagnostic ability of this score and its association with clinical characteristics and age. Results Based on the EIS/AD score, the EIS algorithm was able to clearly discriminate between healthy skin and clinically unaffected skin of children with active AD (area under the curve 0.92, 95% CI 0.85–0.99). It was also able to detect a difference between healthy skin and AD skin when the child did not have active AD. There was no clear association between the EIS/AD score and the severity of AD or sensitisation to the tested allergens. The performance of the algorithm was not affected by age. Conclusions This study shows that EIS can detect skin barrier dysfunction and differentiate skin of children with AD from healthy skin and suggests that EIS may have the ability to predict future AD development

Electrical impedance spectroscopy detects skin barrier dysfunction in childhood atopic dermatitis.

BACKGROUND Skin barrier dysfunction is associated with the development of atopic dermatitis (AD), however methods to assess skin barrier function are limited. We investigated the use of electrical impedance spectroscopy (EIS) to detect skin barrier dysfunction in children with AD of the CARE (Childhood AlleRgy, nutrition, and Environment) cohort. METHODS EIS measurements taken at multiple time points from 4 months to 3-year-old children, who developed AD (n = 66) and those who did not (n = 49) were investigated. Using only the EIS measurement and the AD status, we developed a machine learning algorithm that produces a score (EIS/AD score) which reflects the probability that a given measurement is from a child with active AD. We investigated the diagnostic ability of this score and its association with clinical characteristics and age. RESULTS Based on the EIS/AD score, the EIS algorithm was able to clearly discriminate between healthy skin and clinically unaffected skin of children with active AD (area under the curve 0.92, 95% CI 0.85-0.99). It was also able to detect a difference between healthy skin and AD skin when the child did not have active AD. There was no clear association between the EIS/AD score and the severity of AD or sensitisation to the tested allergens. The performance of the algorithm was not affected by age. CONCLUSIONS This study shows that EIS can detect skin barrier dysfunction and differentiate skin of children with AD from healthy skin and suggests that EIS may have the ability to predict future AD development

The abundance of Ruminococcus bromii is associated with faecal butyrate levels and atopic dermatitis in infancy

Background: Impaired microbial development and decreased levels of short chain fatty acids, particularly butyrate, is suggested to have a role in the development of atopic dermatitis (AD). Methods: Faecal microbiota composition, abundance of selected bacterial groups and fermentation metabolites were compared at 90, 180 and 360 days of life between 27 children who developed AD by age one (AD group), and 39 controls (non-AD group) among the CARE (Childhood AlleRgy, nutrition and Environment) study cohort. Results: Diversity within the Firmicutes and Bacteroidetes phylum in the faecal microbiota was lower in the AD group compared to the non-AD group. Longitudinal analysis showed multiple amplicon sequence variants (ASV) within the same bacterial family to be differentially abundant. Namely, Ruminococcus bromii, a keystone primary starch degrader, and Akkermansia muciniphila, a mucin-utilizer, had lower abundance among the AD group. Children with AD were less likely to have high levels of faecal butyrate at 360 days compared to those without AD (11.5% vs 34.2%). At 360 days, children with high abundance of R. bromii had higher level of butyrate as well as lower proportion of children with AD compared to children with low abundance of R. bromii (11.1-12.5% vs 44.4-52.5%), which was independent of the abundance of the major butyrate producers. Conclusion: Our results suggested that R. bromii and other primary degraders might play an important role in the differences in microbial cross-feeding and metabolite formation between children with and without AD, which may influence the risk of developing the disease. Keywords: atopic dermatitis; butyrate; microbiota; resistant starch; short chain fatty acid

Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78

The pre- and postnatal environment may represent a window of opportunity for allergy and asthma prevention, and the hygiene hypothesis implies that microbial agents may play an important role in this regard. Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis, maternal (prenatal) microbial exposure, and the involvement of Toll-like receptor (TLR) signaling in prenatal protection from asthma. Maternal intranasal exposure to A. lwoffii F78 protected against the development of experimental asthma in the progeny. Maternally, A. lwoffii F78 exposure resulted in a transient increase in lung and serum proinflammatory cytokine production and up-regulation of lung TLR messenger RNA. Conversely, suppression of TLRs was observed in placental tissue. To investigate further, the functional relevance of maternal TLR signaling was tested in TLR2/3/4/7/9−/− knockout mice. The asthma-preventive effect was completely abolished in heterozygous offspring from A. lwoffii F78–treated TLR2/3/4/7/9−/− homozygous mother mice. Furthermore, the mild local and systemic inflammatory response was also absent in these A. lwoffii F78–exposed mothers. These data establish a direct relationship between maternal bacterial exposures, functional maternal TLR signaling, and asthma protection in the progeny

Histamine receptor 2 is required to suppress innate immune responses to bacterial ligands in patients with inflammatory bowel disease

BACKGROUND: Histamine is a key immunoregulatory mediator in immediate-type hypersensitivity reactions and chronic inflammatory responses, in particular histamine suppresses proinflammatory responses to bacterial ligands, through histamine receptor 2 (H2R). The aim of this study was to investigate the effects of histamine and H2R on bacteria-induced inflammatory responses in patients with IBD. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Crohn's disease, patients with ulcerative colitis, and healthy controls. PBMC histamine receptor expression was evaluated by flow cytometry. Cytokine secretion following Toll-like receptor (TLR)-2, TLR-4, TLR-5, or TLR-9 stimulation in the presence or absence of histamine or famotidine (H2R antagonist) was quantified. Biopsy histamine receptor gene expression was evaluated using reverse transcription-polymerase chain reaction. The in vivo role of H2R was evaluated in the T-cell transfer murine colitis model. RESULTS: The percentage of circulating H2R monocytes was significantly reduced in patients with IBD. Histamine effectively suppressed TLR-induced cytokine secretion from healthy volunteer PBMCs but not for PBMCs from patients with IBD. Famotidine reversed this suppressive effect. H1R, H2R, and H4R gene expression was increased in inflamed gastrointestinal mucosa compared with noninflamed mucosa from the same patient and expression levels correlated with proinflammatory cytokine gene expression. Mice receiving lymphocytes from H2R donors, or treated with famotidine, displayed more severe weight loss, higher disease scores and increased numbers of mucosal IFN-γ and IL-17 T cells. CONCLUSION: Patients with IBD display dysregulated expression of histamine receptors, with diminished anti-inflammatory effects associated with H2R signaling. Deliberate manipulation of H2R signaling may suppress excessive TLR responses to bacteria within the gut

The role of specific Toll-like receptors in host recognition of <i>Staphylococcus aureus</i> and in stabledust-mediated protection against allergic asthma

Toll-like Rezeptoren (TLRs) erkennen eine Vielzahl von konservierten mikrobiellen Komponenten. Welche Rezeptoren die zelluläre Erkennung von Gram-positiven Bakterien vermitteln, ist bisher nicht bekannt. Als erstes Ziel dieser Arbeit wurden Mäuse gezüchtet, die für TLR 2, 3, 4, 7 und 9 defizient sind und die immunologische Reaktion dieser Mäuse auf Gram-positive Bakterien und im akuten Asthma untersucht. Es konnte gezeigt werden, dass Knochenmarksmakrophagen das Gram-positive Bakterium Staphylococcus aureus unabhängig von den TLRs 2, 3, 4, 7 und 9, aber abhängig von den bislang wenig beschriebenen TLRs 8 und 13 erkennen. Dabei wurde als aktivierender Ligand die RNA Gram-positiver Bakterien identifiziert. Weiterhin wurde in dieser Arbeit die Rolle von TLRs in der Pathogenese von OVA induziertem Asthma untersucht. Die Analyse der TLR2/3/4/7/9-/- Mäuse in einem Modell akuten Asthmas zeigte, dass abhängig von der Art der Sensitivierung TLRs eine entscheidende Rolle in der Asthmapathogenese und in der Erkennung von Stallstaub, welche dann zum Schutz vor Asthma führt, spielen.Toll-like receptors (TLRs) recognize a broad variety of conserved microbial components. Little is known about the receptors involved in cellular sensing of gram positive bacteria. The primary aim of this thesis was to generate mice deficient in TLRs 2, 3, 4, 7 and 9 and test the immunological responses of those mice to gram positive bacterial infection or asthma. Data obtained using bone marrow derived macrophages from those mice showed that recognition of the gram positive bacterium Staphylococcus aureus was independent of TLRs 2, 3, 4, 7 and 9 but dependent on the poorly described TLRs 8 and 13. Furthermore RNA from gram positive bacteria was identified to be the activating ligand of these TLRs. In a further study the role of TLRs in the pathogenesis of OVA induced asthma was analyzed. Analysis of TLR2/3/4/7/9-/- mice in a model of acute asthma outlined, depending of the mode of sensitization, an important role for TLRs in the pathogenesis of asthma and recognition of stable dust to elicit subsequent protection

The surface-associated exopolysaccharide of bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses