SEARCH

SEARCH BY CITATION

Keywords:

  • allergy;
  • animal model;
  • asthma;
  • farming effect;
  • prevention;
  • proof-of-concept

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

Background

Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation.

Methods

We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to Ssciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co-culture with autologous T cells.

Results

The allergo-protective properties of S. sciuri W620 were proven in a TH2-driven OVA model as well as in a mixed TH1/TH2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell TH1/TH2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in moDC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported TH1 cytokine release by co-cultured T cells.

Conclusion and clinical relevance

Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.

Abbreviations
BAL

bronchoalveolar lavage

CFU

colony-forming unit

CI

confidence interval

DC

dendritic cells

GABRIELA

A Multidisciplinary Study to Identify the Genetic and Environmental Causes of Asthma in the European Community – Advanced Studies

HDM

house dust mite

IL

interleukin

LAB

lactic acid bacteria

moDC

monocyte-derived dendritic cells

NOD

nucleotide-binding oligomerization domain

OR

odds ratio

OVA

ovalbumin

PAS

periodic acid-Schiff

PBS

phosphate buffer saline

PCR

polymerase chain reaction

PRR

pattern-recognition receptor

SEM

standard error of the mean

SSCP

single-strand conformation polymorphism

TLR

Toll-like receptor

Studies conducted in traditional farming sites identified dust collected in this environment to harbor a plethora of microorganisms, which might provide protection against allergies and asthma. In the context of exposure to environmental microorganisms, the GABRIELA study (A Multidisciplinary Study to Identify the Genetic and Environmental Causes of Asthma in the European Community – Advanced Studies) located several hot spots with a protective potential against allergic outcomes [1]. In-depth analyses as presented by Ege et al. [2] identified a cluster of bacterial species to be inversely associated with atopy and asthma. Out of this cluster, we selected Gram-positive Staphylococcus sciuri W620 for an experimental proof-of-concept to demonstrate that the preceding analytical approach presented by Ege et al. [2]. is suitable to identify microbial species with actual allergo-protective properties. Based on protocols initiating an allergic airway inflammation either with ovalbumin (OVA) or house dust mite (HDM), we employed in vivo models of murine airway inflammation to prove if S. sciuri W620 is capable to prevent experimental asthma under TH2- and TH1/TH2-driven conditions. As a Gram-positive bacterium, it may provide protection by pathways different to those described previously for the farm-derived Gram-negative isolate A. lwoffii F78 [3, 4]. We aimed to characterize cytokine patterns in response to S. sciuri W620 in vivo and in vitro to get first hints on the underlying mechanisms of immune cell activation and maturation.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

Detection, isolation, and propagation of S. sciuri W620

For detection of S. sciuri W620, PCR-SSCP was performed as described previously [5]. In addition, PCR targeting the mecA gene as a specific marker was performed according to Couto et al. [6]. Further details on S. sciuri W620 detection, isolation, and propagation are given in the method section of the online supplement.

Animal experiments

Mouse models of allergic airway inflammation

Female Balb/c mice (6–8 weeks old) were housed in a pathogen-free facility with individually ventilated cages and received OVA- and HDM-free diet. All animal experiments were approved by the local authorities. At least 6–8 animals were used per group. In the OVA model, mice were sensitized to OVA on days 0, 7, and 14 by subcutaneous (s.c.) injections of 10 μg OVA (grade VI; Sigma, Steinheim, Germany) in 200 μl PBS. Sham-treated groups received 200 μl PBS. The sensitization phase was followed on days 26, 27, and 28 by 20-min aerosol applications of 1% OVA or sham PBS, as described previously [7] (Fig. 1A).

image

Figure 1. Sensitization and challenge protocols used for murine experimental asthma induction. (A) OVA model with subcutaneous sensitization and aerosol challenge. (B) HDM model with intranasal sensitization and challenge.

Download figure to PowerPoint

In the HDM model, mice were exposed intranasally (i.n.) to 100 μg Dermatophagoides pteronyssinus extract (Allergopharma, Hamburg, Germany) dissolved in 25 μl PBS on days 0, 7, 14, and 21; control animals received 25 μl PBS (Fig. 1B).

Treatment with S. sciuri W620

Anesthetized mice were treated i.n. with 108 colony-forming units (CFU) of lyophilized S. sciuri W620 suspended in 25 μl PBS in the HDM model and 50 μl in the OVA model, respectively, or 25 μl PBS 3 times per week, starting 10 days before the first sensitization and continuing over the whole sensitization and challenge process (Fig. 1A and B).

Characterization of the allergic phenotype

To characterize the allergic phenotype, the following parameters were analyzed: (i) differential cell counts in bronchoalveolar lavage (BAL), (ii) number of mucus-producing Goblet cells and inflammation score in lung histology, (iii) airway hyperresponsiveness to methacholine (Mch), (iv) serum immunoglobulins, (v) cytokines in BAL fluid and in restimulated lymph node cell supernatants after restimulation, and (vi) frequency of CD4 + Foxp3 + CD25 +  T regulatory (Treg) cells in lung homogenates. All these methods are described in detail in the online supplement.

In vitro experiments in human cells

Transient transfection of HEK293 cells

HEK293 cells were transiently transfected with various pattern-recognition receptor (PRR) expression plasmids as described before [3]. More details are given in the online supplement.

Generation and stimulation of moDC

Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood of healthy donors as described previously [8]. Subsequently, monocytes were isolated by counterflow elutriation centrifugation [9]; monocyte-derived DCs were then prepared as described by Sallusto and Lanzavecchia [10]. For further details, see online supplement.

Expression of costimulatory molecules on human moDC

After 20 h of incubation with the indicated stimuli, cells were harvested and further analyses were carried out by using FACS analysis, real-time quantitative (RT) PCR, and ELISA. Further details are described in the online supplement.

Co-culture of moDCs with autologous naïve T cells

Co-cultures of moDCs with autologous naïve CD4 +  T cells were performed as previously described [11]. Cytokine protein and mRNA expression was measured as readout for T-cell activation. Further details are given in the online supplement.

Statistical analyses

Data analysis of dust samples was carried out using stata 10.1 (StataCorp, College Station, TX, USA). Detection by PCR was used as a dichotomous variable split at the detection limit. The arbitrary density units of the SSCP analysis were used as log-transformed variables. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by logistic regression for analyses of associations between SSCP and PCR products with asthma.

Data analyses of animal experiments and human dendritic cell experiments were performed with GraphPad Prism® (GraphPad Software Inc., La Jolla, CA, USA). All numeric data are expressed as means ± SEM and analyzed for significance by using one-way anova and subsequent Tukey analysis. Differences were considered significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

Exposure to S. sciuri W620 is significantly associated with asthma protection in farm children

In a population-based survey and using PCR-SSCP, we have previously identified several bacteria as candidates for asthma-protective agents [2]. These included a cluster containing Staphylococcaceae, which was associated with a significantly lower asthma risk [2]. This cluster contained among others the species Staphylococcus sciuri, which was confirmed by sequencing in triplets. Furthermore, a specific PCR targeting the mecA gene, which is considered specific for the species S. sciuri, revealed a similarly strong association with asthma protection (OR = 0.55 [0.22–1.34]) although barely missing statistical significance (P = 0.188), due to a reduced sample size (n = 371). The association between detection of S. sciuri by PCR-SSCP was significant (OR = 1.29 [1.03–1.61], P = 0.025) thereby cross-validating both methods. S. sciuri was detected in significantly higher amounts in mattress dust of farm children compared to nonfarm children (OR = 9.69 [6.04–15.54], P < 0.001).

Intranasal exposure to S. sciuri W620 in the OVA-induced mouse model of airway inflammation resulted in a decrease of hallmarks of a TH2-driven asthmatic phenotype

To prove whether S. sciuri W620 is able to abolish a TH2-driven airway inflammation in an experimental setup, we employed a well-established mouse model of experimental asthma based on a subcutaneous OVA-sensitization protocol (Fig. 1A). Mice were treated with S. sciuri W620 prior to and during sensitization and challenge. We found total BAL cell counts, eosinophils, and lymphocyte numbers as major hallmarks of airway inflammation to be significantly decreased in S. sciuri W620-treated OVA-sensitized/challenged animals in comparison with the control group. Macrophage and neutrophil numbers were not affected by intranasal exposure to S. sciuri W620 (Fig. 2A).

image

Figure 2. Intranasal application of S. sciuri W620 abolishes hallmarks of acute airway inflammation in a murine OVA-induced model of experimental asthma. (A) Total and differential cell counts in the BAL fluid of BALB/c mice subjected to an OVA s.c. adjuvant free (Protocol 1A); (B) Goblet cells and inflammation score in lung tissues; (C) Airway inflammation and mucus producing Goblet cells in PAS-stained histologic lung section. (D) Serum OVA-specific immunoglobulins IgE, IgG1, and IgG2a; (E) Cytokine production in the BAL fluid measured by ELISA; (F) Cytokine production by lymph node cells after 72 h re-stimulation with OVA measured by ELISA; (G) Airway hyperresponsiveness measured by head-out body plethysmography. *P < 0.05, **P < 0.01, ***P < 0.001. Shown are means + SEM.

Download figure to PowerPoint

Evaluation of PAS-stained lung sections revealed a significantly lower peribronchial/perivascular inflammation and reduced Goblet cell hyperplasia in animals treated with S. sciuri W620 compared to the OVA control group (Fig. 2B). As a functional parameter, airway hyperresponsiveness (AHR) was improved in the S. sciuri W620-treated group (Fig. 2C).

Intranasal S. sciuri W620 treatment was associated with significantly lower OVA IgE and OVA IgG1 serum levels compared to untreated animals while OVA IgG2a levels did not differ between the OVA and the S. sciuri/OVA groups (Fig. 2D).

Furthermore, we analyzed T-cell signature cytokine production in BAL and OVA re-stimulated lymph node cell supernatants. In BAL fluid, we found only IL-5, but not IL-10 and IFN-γ (Fig. 2E) to be significantly reduced following S. sciuri W620 exposure, whereas all these cytokine levels were ameliorated in lymph node cell supernatants (Fig. 2F).

Staphylococcus sciuri W620 prevents the asthma phenotype in a murine HDM-induced allergic airway inflammation model

Our data supported an abolishment of selectively TH2-driven airway inflammation by i.n. treatment with S. sciuri W620 in the OVA model. To prove if S. sciuri W620 is also capable of preventing a mixed airway inflammation in mice, we employed a house dust mite (HDM) model, which in parallel displays neutrophilic and eosinophilic airway inflammation. According to the protocol given in Fig. 1B, mice were treated with S. sciuri W620 prior and during HDM extract application.

We observed a significantly reduced BAL cell numbers in S. sciuri W620-exposed allergen-challenged mice as compared to the control group (Fig. 3A, left panel). BAL differential cell counts indicated significantly lower numbers mainly of neutrophils (24 h after last challenge) and eosinophils (48 h after last challenge) (Fig. 3A).

image

Figure 3. Intranasal application of S. sciuri W620 abolishes hallmarks of acute airway inflammation in a murine HDM model of experimental asthma. (A) Total and differential cell counts in the BAL of BALB/c mice subjected to a HDM model of experimental asthma, both 24 and 48 hours after the last challenge; (B) Goblet cells and inflammation score in lung tissues. (C) Airway inflammation and goblet cell producing cells in PAS-stained histologic lung section. (D) Serum HDM-specific immunoglobulins IgG1 and IgG2a. (E) Cytokine production by lymph node cells after 72 h re-stimulation with HDM. (F) Protein levels of CCL11 (eotaxin) in lung homogenates (48 h after last challenge) and CXCL1 (KC) in BAL (24 h after last challenge). (G) Percentages of CD4 + CD25 + Foxp3 +  regulatory T cells as determined by flow cytometry of homogenized lungs *P < 0.05, **P < 0.01, ***P < 0.001. Shown are means + SEM.

Download figure to PowerPoint

Additionally, there were significantly lower numbers of PAS-positive mucus-producing cells in the airways of S. sciuri W620-treated allergen-challenged mice compared with HDM control animals (Fig. 3B).

Even though S. sciuri W620 treatment appeared to suppress HDM-induced airway inflammation, we could not observe any significant changes in HDM-specific IgG1 and IgG2a serum levels as shown in Fig. 3C. HDM-specific IgE serum levels were not detectable in any group.

Similar to the experiments within the OVA model, we analyzed T-cell cytokines in cell supernatants from the draining lymph nodes. To monitor a broad spectrum of T-cell-derived cytokines, we analyzed a panel of TH1 (IFN-γ; IL-17A), TH2 (IL-4, IL-5, IL-9, IL-13), and Treg (IL-10) cytokines in re-stimulated T cells and observed an increase of these cytokines in the sham-treated HDM-administered animals, whereas mice treated with S. sciuri W620 were characterized by a significant reduction of all of these cytokines (Fig. 3D).

As eosinophils and neutrophils were found to be significantly reduced in the BAL fluid of S. sciuri W620-treated allergen-challenged mice, we also analyzed whether the chemokines CXCL1 (KC) and eotaxin-1 (CCL11) playing an important role in the recruitment of those cells into the lung are also affected. We observed markedly lower mRNA (data not shown) and protein levels of the two chemokines in S. sciuri W620-treated allergen-challenged mice compared to the sham-treated allergen-challenged group as depicted in Fig. 3F.

Microbial components have been proposed to drive suppression of allergic immune responses via induction of counteracting TH1 as well as Treg cell responses. Therefore, we also measured CD4 + Foxp3 + CD25 +  Treg frequencies in lung homogenates. As shown in Fig. 3G, S. sciuri W620 treatment did not influence Treg frequencies in the lung in comparison with the sham-treated allergen-challenged group.

S. sciuri W620 activates TLR2 and NOD2 in transfected HEK293 cells

Studies on the activation of innate immune responses might further elucidate mechanisms preceding altered T-cell responses to S. sciuri W620 exposure. To explore S. sciuri W620 signaling through specific pattern-recognition receptors (PRR), we employed a well-established assay of transiently transfected HEK cells. Incubation of differentially transfected HEK cells with S. sciuri W620 resulted in a dose-dependent induction of Toll-like-receptor (TLR)2 activity comparable to that following TLR2 agonist Pam3CysSK4 exposure. In addition, intracellular NOD2 was activated by S. sciuri W620. There was no activation of HEK cells through TLR4 and NOD1 (Table 1 and Fig. S1).

Table 1. Innate immune receptors addressed by S. sciuri W620 in a HEK293 cell transfection system
PRRTLR2TLR4NOD1NOD2
  1. a

    CXCL-8-inducing capacity in HEK293 cells transiently transfected with indicated PRR (no activity has been detected with any other TLR). For detailed data, see Fig. S1.

Activationa++

Maturation of human moDC is initiated by S. sciuri W620

As S. sciuri W620 triggered signaling via innate immune receptors, it was next tested whether stimulation of moDC with the bacterium leads to maturation of these cells as indicated by up-regulation of surface expression of co-stimulatory molecules. Incubation of moDC with S. sciuri W620 led to up-regulation of CD40, CD86, and CD80. In addition, moDC stimulated with the bacterium showed a higher expression of the antigen-presenting major histocompatibility complex (MHC) class II (Fig. 4).

image

Figure 4. S. sciuri W620 induces maturation of moDCs. MoDCs were stimulated with 107 cfu/ml S. sciuri W620 or left untreated (control). After 20 h, surface expression of CD40, CD80, CD86, and MHCII was analyzed by flow cytometry. Gray filled histograms show isotype control antibody binding, black dashed line surface expression on untreated control cells, and black lines expression after S. sciuri treatment. Representative FACS plots of n ≥ 8 individual analyses are shown.

Download figure to PowerPoint

S. sciuri W620 induces IL-23 but not IL-12p70 release by human dendritic cells due to lack of IL-12p35 mRNA induction

As S. sciuri W620 exposure to dendritic cells led to an activation and maturation of moDC, we aimed to analyze pro-inflammatory cytokine expression following bacterial activation by S. sciuri W620. We found IL-6, CXCL-8, and TNF-α production to be up-regulated after S. sciuri W620 treatment (data not shown). No release of IL-12p70 could be observed, whereas IL-23 was detected. As IL-12p70 is a heterodimeric cytokine consisting of the two subunits p35 and p40, mRNA expression of these subunits was analyzed separately by real-time PCR. Only the mRNA of IL-12p40 was detectable (Fig. 5). S. sciuri W620 induced transcription of IL-23p19 mRNA in moDC; hence, both subunits of IL-23, that is, IL23p19 and the IL12p40, were transcribed.

image

Figure 5. S. sciuri W620 induces IL-12p40 and IL-23p19 mRNA in MoDCs but not IL-12p35 and leads to release of IL-23 but not IL-12p70. (A) MoDCs were stimulated with LPS [100 ng/ml] or S. sciuri W620 [10cfu/ml] for 0, 3, 6, and 12 h before mRNA was isolated. Expression of IL-12p35, p40, and IL-23p19 mRNA was calculated relative to HPRT expression by real-time PCR. Shown are results of one of five experiments. (B) MoDCs were left untreated (control) or stimulated with LPS or S. sciuri W620 at indicated concentrations for 20 h. IL-12p70 and IL-23 content was measured in the supernatants by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, shown are means + SD.

Download figure to PowerPoint

Co-culture of moDC with autologous T cells selectively induced TH1 cytokine release

We finally investigated the influence of S. sciuri W620-exposed moDC on the activation of autologous naïve T cells. As depicted in Table 2, no induction of IL-4 and IL-10 was observed. However, S. sciuri W620-exposed moDC were able to induce the release of IFN-γ by T cells in comparable extend to LPS-stimulated moDC.

Table 2. Cytokine production by human moDC/naïve T-cell (TC) co-cultures in absence and presence of S. sciuri W620 and LPS (as control)a
 moDCsmoDCs + TC
MediumLPS [100 ng/ml]S. sciuri W620 [10cfu/ml]MediumLPSS. sciuri W620 [10cfu/ml]
  1. a

    All data are depicted as mean ± SEM in [ng/ml] of at least 5 independent experiments.

IFN-γ0.01 ± 0.010.05 ± 0.030.17 ± 0.110.40 ± 0.123.07 ± 1.452.91 ± 1.07
IL-100.02 ± 0.000.01 ± 0.000.00 ± 0.000.02 ± 0.010.16 ± 0.070.10 ± 0.04
IL-40.00 ± 0.000.00 ± 0.000.01 ± 0.010.00 ± 0.010.09 ± 0.050.06 ± 0.03

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

Our proof-of-concept experiments clearly demonstrate that screening of farm-derived dust samples as conducted by Ege et al. [2] is a powerful method to identify bacterial species that harbor such properties. S. sciuri W620 is the first asthma-protective Gram-positive isolate from farm-derived dusts that could be identified by this structured approach.

Exemplary for other microorganisms that were found to be associated with asthma protection in these dust sample analyses, S. sciuri W620 was investigated in two murine models of acute allergic airway inflammation. Using the OVA model, we emphasized possible preventive effects of S. sciuri W620 on the development of a distinctly TH2-driven allergic inflammation. The HDM model was set up to study asthma prevention under a mixed TH1/TH2 condition characterized by an airway inflammation with a paralleled neutrophil and eosinophil influx into the lung. In both S. sciuri W620-exposed asthma phenotypes, a significantly diminished allergic airway inflammation characterized by a clear decrease of the respective inflammatory cells in the lung accompanied by a significant reduction of Goblet cells was achieved by treatment with S. sciuri W620 in comparison with sham-treated control animals. Additionally, in the HDM model, chemokines that attract eosinophils and neutrophils such as CCL11 (eotaxin-1) and CXCL1 (KC) were reduced in the lungs of S. sciuri W620-treated mice.

Previously, we have described Acinetobacter lwoffii F78 as a Gram-negative isolate from dust samples to protect against experimental asthma when applied intranasally in in vivo models with pre- and postnatal exposure [3, 4]. In vitro experiments with A. lwoffii F78-exposed human moDCs revealed an activation of IL-12 production and a subsequent polarization of naive T cells toward a TH1 phenotype as shown by a strong IFNγ production in autologous naive T cells co-cultured with A. lwoffii F78-exposed moDCs. These effects were mainly driven by A. lwoffii F78 LPS indicating that early exposure to Gram-negative bacteria might protect against retardation of immune maturation and persistence of the developing immune system within the TH2-driven prenatal milieu [11]. In analogy to these A. lwoffii F78 experiments, treatment of human moDCs with S. sciuri W620 also led to a maturation of dendritic cells in this case via TLR2/NOD2-mediated signaling processes. Contrary, no release of IL-12 was observed after incubation of moDCs with the Gram-positive bacterium. mRNA analyses revealed that the IL-12p35 gene was not transcribed in dendritic cells incubated with S. sciuri W620 while mRNA transcripts for IL-12p40 as well as IL23p19 were detectable. In consequence, IL-23, a heterodimer assembled of the IL-12p40 and IL-23p19 subunits, could be detected in supernatants from S. sciuri W620-exposed cells while production of IL-12 was absent. Actually, it was previously described that binding and signaling through TLR2 may inhibit IL-12 production while IL-23 release was increased [12]. IL-12 plays a major role in mounting appropriate immune responses to pathogens and was characterized to have a central role in the development of TH1 responses [13, 14]. Hence, missing IL-12 production might explain incomplete TH1 maturation and diminished TH1 cytokine production in animal models. However, in vitro co-culture experiments revealed that naive T cells are instructed to produce IFN-γ possibly due to IL-23 release by moDCs. Whereas it is known that in vivo IL-23 preferentially acts on memory CD4+  T cells to produce IFN-γ [15], under in vitro conditions this might be different. In line with the in vitro studies, in both in vivo experiments, no IL-12 production was detectable in lungs and lymph nodes of S. sciuri W620-treated animals, whereas IL-23 production tended to be increased without reaching statistical significance (data not shown). Nevertheless, in both in vivo models, no increase of IFN-γ could be observed in the BAL and the lung-draining lymph nodes of S. sciuri W620-treated animals following local allergen challenge. This implies that S. sciuri W620 seems to act in vivo via a rather generalized suppression of T-cell activities at least during the effector phase, which is also supported by the observation of missing Treg induction.

Our experiments demonstrated that continuous S. sciuri W620 exposure is capable to abrogate inflammatory conditions in the lung. So far, out of Gram-positive bacteria, probiotic lactic acid bacteria (LAB) were shown to provide anti-inflammatory effects mainly in gut [16] but also in the lung when orally given [17, 18]. Recently, we demonstrated that intranasal application of Lactococcus lactis G121 isolated from farm-derived dust samples abolished the asthmatic phenotype in OVA-sensitized mice [3]. Similar to treatment with S. sciuri W620, we observed a decrease of lymph node cell-derived cytokine production after application of L. lactis G121 to OVA-sensitized mice (H. Harb, H. Garn, P. I. Pfefferle, unpublished data).

Taken together, observations of this and our previous studies suggest that protection against airway inflammation by Gram-positive and Gram-negative bacteria is achieved by different modes of action. The farm effect is mainly based on the microbial diversity found in rural farming sites [1]. Combination of different mechanisms of action might enhance protective effects as signaling through multiple pathways may balance the immune response toward tolerance. The screening system presented by Ege et al. [2]. may offer an array of candidates to be analyzed in single-strain experiments for mechanistic pathways and in mixed application for further clinical treatment of allergic diseases.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Sophia Bernhardt, Lydia Lerch, Thomas Ruppersberg, and Alexandra Fischer for excellent technical assistance. This work was supported by the DFG TR22 ‘Allergic responses of the lung’ (A1 von Mutius/Bauer; A2 Heine/Holst; Z2 Garn).

Author contributions

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information

SH, HHb, HHe, HR, HG, and PP were involved in the design of the study; ME, EvM, MM, JM, and JB were involved in epidemiology; SH, HHb, ZM, PP, and HG performed animal experiments; KS and HHe carried out in vitro studies; all authors were involved in the preparation of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information
  • 1
    Ege MJ, Mayer M, Normand AC, Genuneit J, Cookson WO, Braun-Fahrländer C et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011;364:701709.
  • 2
    Ege MJ, Mayer M, Schwaiger K, Mattes J, Pershagen G, van Hage M et al. Environmental bacteria and childhood asthma. Allergy 2012;67:15651571.
  • 3
    Debarry J, Garn H, Hanuszkiewicz A, Dickgreber N, Blümer N, von Mutius E et al. Acinetobacter lwoffii and Lactococcus lactis strains isolated from farm cowsheds possess strong allergy-protective properties. J Allergy Clin Immunol 2007;119:15141521.
  • 4
    Conrad ML, Ferstl R, Teich R, Brand S, Blümer N, Yildirim AO et al. Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78. J Exp Med 2009;206:28692877.
  • 5
    Korthals M, Ege MJ, Tebbe CC, von Mutius E, Bauer J. Application of PCR-SSCP for molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust. J Microbiol Methods 2008;73:4956.
  • 6
    Couto I, Sanches IS, Sá-Leão R, de Lencastre H. Molecular characterization of Staphylococcus sciuri strains isolated from humans. J Clin Microbiol 2000;38:11361143.
  • 7
    Conrad ML, Yildirim AO, Sonar SS, Kiliç A, Sudowe S, Lunow M et al. Comparison of adjuvant and adjuvant-free murine experimental asthma models. Clin Exp Allergy 2009;39:12461254.
  • 8
    Boyum A. Separation of White Blood Cells. Nature 1964;204:793794.
  • 9
    Turpin J, Hester JP, Hersh EM, Lopez-Berestein G. Centrifugal elutriation as a method for isolation of large numbers of functionally intact human peripheral blood monocytes. J Clin Apher 1986;3:111118.
  • 10
    Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994;179:11091118.
  • 11
    Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010;65:690697.
  • 12
    Gerosa F, Baldani-Guerra B, Lyakh LA, Batoni G, Esin S, Winkler-Pickett RT et al. Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J Exp Med 2008;205:14471461.
  • 13
    Manetti L, Parronchi P, Giudizi MG, Piccinni MP, Maggi E, Trinchieri G et al. Natural killer cell stimulatory factor (interleukin-12 [IL-12]) induces T helper type 1 (TH1)-specific immune responses and inhibits the development of IL-4-producing TH cells. J Exp Med 1993;177:11991204.
  • 14
    Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol 1998;70:83243.
  • 15
    Lankford CS, Frucht DM. A unique role for IL-23 in promoting cellular immunity. J Leukoc Biol 2003;73:4956.
  • 16
    Calder PC, Albers R, Antoine JM, Blum S, Bourdet-Sicard R, Ferns GA et al. Inflammatory disease processes and interactions with nutrition. Br J Nutr 2009;101(Suppl 1):S1S45.
  • 17
    Osborn DA, Sinn JK. Probiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database Syst Rev 2007;4:CD006475.
  • 18
    Borchers AT, Selmi C, Meyers FJ, Keen CL, Gershwin ME. Probiotics and immunity. J Gastroenterol 2009;44:2646.

Supporting Information

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author contributions
  8. Conflict of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
all12094-sup-0001-Data S1.docxWord document335KSupplement S1. Methods and results

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.