University of Groningen

Effects of environmental exposures on asthma phenotypes in the mouseBlacquière, Margareta Johanna

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Chapter 7Farm dust exposure and development of non-

eosinophilic airway inflammation in mice: the role of epithelium, macrophages

and B cell follicles

MJ Blacquière

DS Postma, W Timens

M Geerlings, A Lech

A Bufe, O Holst

MN Hylkema

AbstrAct

introduction Farming life can on the one hand protect against development of allergies and allergic asthma, but on the other hand induce airway symptoms like dyspnea, cough and wheezing in non-allergic individuals. We recently showed that farm dust exposure decreases house dust mite (HDM)-induced IgE and eosinophilic airway inflammation and simultaneously induces non-allergic airway inflammation.Aim To investigate mechanisms underlying the observed decreased allergic airway in-flammation and simultaneously increased, possibly TH17 mediated, non-allergic airway inflammation.methods Lungs of mice previously exposed to dust from cowsheds were studied in detail for Toll-like receptor (TLR)2 and TLR4 expression in epithelium, thymic stromal lymphopoietin (TSLP) levels, the number of alternatively activated macrophages and the composition of infiltrates. Mice were intranasally exposed 4 times/week for 5 weeks to phosphate buffered saline (PBS) or farm dust (1 mg/ml, 50 µl/day, collected from five different farms in North Germany), followed by PBS or HDM (2.5 mg/ml, 10 µl/day). results Farm dust exposed mice had lower TLR2 and TLR4 expression in epithelium and TSLP levels in lung tissue than HDM exposed mice. Expression of YM1 by alternatively ac-tivated macrophages was lower in farm dust exposed mice as well. The infiltrates in lung tissue from dust exposed mice consisted of B cells and follicular dendritic cells and lgG1 levels in serum were higher in farm dust exposed mice than in HDM exposed mice.conclusions Downregulation of TLR2 and TLR4 expression in epithelium may contribute to the observed dust-induced, non-allergic asthma phenotype, since downregulation of TLR expression associated with lower levels of TSLP. Lack of TSLP may have prevented induction of allergic sensitisation and airway inflammation in farm dust exposed mice. Additionally, we observed formation of B-cell follicles in dust exposed mice, which could be caused by the observed higher numbers of TH17 cells in these mice.

introduction

Asthma is defined as a chronic inflammato-ry disease of the airways, which is charac-terized by airway hyperresponsiveness and leads to recurrent episodes of wheezing, breathlessness, chest tightness and coug-hing (Global INitiative for Asthma, GINA). Many studies have focussed on allergic asthma, which is characterized by eosi-nophilic inflammation, a TH2 type immune response and allergen specific IgE produc-tion. However, a considerable proportion of asthma, sometimes even more than 50%, is not attributable to allergic sensitisation 1. Although not much research has focus-sed on non-allergic asthma specifically, it is nowadays thought that non-allergic asthma does not involve an allergen speci-fic IgE response, tends to display neutrop-hilic instead of eosinophilic inflammation 2-4, is frequently clinically steroid resistant 2,5 and often more severe.Several studies report that the incidence of asthma has increased over the past deca-des worldwide 6-10, especially in industria-lized western countries with increased hy-giene standards. However it is not known whether this increase is attributable to

allergic or non-allergic asthma.. A hypo-thesis explaining the increase in asthma prevalence is the hygiene hypothesis, sta-ting that lack of microbial exposure early in life induces a lack of protection against allergies and (allergic) asthma later in life. Epidemiological studies investigating the effect of living in a farming environment, which causes higher microbial exposure than urban living environments, indeed have shown that farming life may confer protection against allergies and allergic asthma in childhood 11-13. However, far-mers and agricultural workers exposed to farm dust less likely had allergies, but ne-vertheless had respiratory symptoms such as shortness of breath, cough and whee-zing 14. This indicates that farm dust expo-sure may protect against allergies, but can induce non-allergic asthma. Indeed, we found in our animal model of HDM-indu-ced allergic airway inflammation, that farm dust exposure decreases allergic airway inflammation (eosinophilic inflammation and TH2 cytokines in lung tissue, total and HDM-specific IgE in serum) but simultane-ously induced a non-allergic type of airway

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inflammation (large inflammatory infiltra-tes, TH17 cells and associated cytokines) and methacholine (MCh)responsiveness. The mechanisms underlying the decreased allergic airway inflammation and simulta-neously increased (possibly TH17 mediat-ed) non-allergic airway inflammation in our previous study are yet unknown. Recently it was shown that Toll-like receptor (TLR)4 in epithelium is essential for HDM induced allergic airway inflammation 15. Signal-ling through TLR4 resulted in recruitment and activation of dendritic cells, which can skew naïve T cells to differentiate towards the TH2 type, thereby inducing allergic air-way inflammation.

To gain more insight into the mechanisms involved in down regulation of allergic air-way inflammation by farm dust exposure, we studied TLR2 and TLR4 expression in airway epithelium from dust exposed mice. Little is known about the characteristics of non-allergic airway inflammation in non-allergic asthma patients. Since our farm dust exposed mice provide an opportunity to study non-allergic asthma in more de-tail, we aimed to better characterize the airway inflammation in this setting. There-fore, we analysed the cellular composition of farm dust induced infiltrates as well as the antibody levels in these mice in more detail.

mAteriAls And methods

Antigens

Dust was collected from five farms in North Germany and extracted according to the published protocol 16. Hereafter, dust ex-tract is referred to as ‘dust’. Dust expo-sure: 1mg/ml PBS, 50 μl/day.House dust mite (HDM) consisted of crushed whole bodies from dermatopha-goides pteronyssinus (Greer Laboratories, Lenoir, USA). HDM exposure: 2.5 mg/ml PBS, 10 μl/day.

study design

To induce allergic airway inflammation, 10-week old female BALB/c mice were anesthetized with isoflurane and intrana-sally exposed to HDM, or phosphate buff-ered saline (PBS) as a control. Mice were exposed four times per week, during five

consecutive weeks.To investigate the protective effect of dust exposure, mice were anesthetized and in-tranasally exposed to dust, or PBS as a con-trol, 1 min before every exposure to HDM (or PBS) (figure 1). Experimental groups: PBS+PBS exposure (n=8), PBS+HDM ex-posure (n=10), dust+PBS exposure (n=9), dust+HDM exposure (n=10). Twenty-four hours after the last allergen exposure MCh responsiveness was assessed as described previously 17. All animal protocols were approved by the local Committee on Animal Experimenta-tion and were performed under strict gov-ernmental and international guidelines on animal experimentation.

tissue collection Twenty-four hours after the MCh assess-ment, mice were anaesthetized and sacri-ficed. Blood was taken by a heart punction. The two smallest right lung lobes were snap frozen; the remaining lobes were carefully inflated with 0.9 ml 50% Tissue Tek, O.C.T. (Sacura, Zoeterwoude, The Netherlands) in PBS. The left lung lobe was formalin fixed and embedded in paraffin, the two smaller right lung lobes were snap frozen and stored at -80ºC until use.

meAsurement of thymic stromAl lym-phopoietin (tslp) in lung tissue

Snap frozen lung tissue was homogenized and TSLP in lung tissue was measured by Enzyme Linked Immuno Sorbent Assay (ELISA) as described by the manufacturer (R&D systems, Minneapolis, USA).

meAsurement of igg1, igg2A And igm in serum

IgG1, IgG2a and IgM levels in serum were measured by ELISA. Flat bottomed 96-wells plates were coated overnight with anti-mouse IgG1 (BD Biosciences, San Jose, USA), a monoclonal anti mouse Ig-G2a antibody (BD Biosciences, OPTEIA kit) or goat anti mouse Ig (Southern Biotech Associates (SBA), Birmingham, USA) re-spectively. Plates were blocked with assay diluents (IgG1 and IgG2a ELISA) or 0.2% ELK/PBS (IgM ELISA). Serum samples were added and incubated for 2h. A mono-clonal anti-mouse IgG1BIO antibody (BD Biosciences), a monoclonal anti-mouse

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IgG2a antibody (BD Biosciences, OPTEIA kit) or a polyclonal goat anti-mouse IgMBIO antibody (SBA) were added and incubated for 1h (IgG1 and IgG2a ELISA) or 2h (IgM ELISA). Horseradish peroxidase conjugat-ed streptavidin (DAKO, Glostrup, Denmark (IgG1 ELISA) or BD Biosciences, OPTEIA kit (IgG2a ELISA)) or avidin-biotin com-plex (DAKO (IgM ELISA)) was added for 30 min. Plates were developed using tetra methyl benzidine (TMB) substrate (Sigma Aldrich, St Louis, USA), stopped and opti-cal densities were read at 450 nm using a Varioscan ELISA reader (Thermo Scientific, Waltham, USA).

histology And immunohistochemistry

To determine B cells, dendritic cells, fol-licular dendritic cells (fDC), alternatively activated macrophages, TLR2 and TLR4, 4 µm cryosections of lung tissue were stained with a rat anti CD19 antibody (BD Biosciences), a rat anti mouse CD11cPE an-tibody (BD Biosciences ), a rat anti mouse fDCm2 antibody (BD Biosciences), a goat anti mouse YM1BIO antibody (R&D systems, Minneapolis, USA), a goat anti mouse TLR2 or TLR4 antibody (SantaCruz, Santa Cruz, USA) respectively. Expression of TLR2 and TLR4 in epithelium was scored visually for each airway separately (1=low expression, 2=intermediate expression, or 3=high ex-pression) and the mean expression for all airways in the total lung section is shown. Since B cells in infiltrates are tightly packed together, individual cells were difficult to discern and therefore the volume percent-age of B cells in infiltrates was calculated

by morphometric analysis using Leica Qwin image analysis software (Leica Microsys-tems, Wetzlar, Germany). Alternatively ac-tivated macrophages were counted manu-ally in whole lung sections and corrected for the total area of lung section.

stAtisticAl AnAlysis

To determine 1) the effect of HDM expo-sure, 2) the effect of dust exposure, and 3) the interaction of the effect of dust exposure on the effect of HDM exposure, we performed a multiple linear regression analysis (SPSS 14.0 software, SPSS Inc). When residuals were not normally distrib-uted, appropriate log10 or 1/x transforma-tion was performed. A significant HDM ef-fect means that HDM exposed mice differ from PBS exposed mice. A significant dust effect means that dust+PBS exposed mice differ from PBS exposed mice. A signifi-cant interaction between the effect of dust exposure and the effect of HDM exposure means that the effect of HDM exposure is different in dust+HDM exposed mice com-pared with PBS+HDM exposed mice.

results

fArm dust exposure decreAses tlr2 And tlr4 expression in epithelium

Figure 1 shows that HDM exposed mice had higher expression of TLR2 and com-parable expression of TLR4 in epithelium compared with PBS exposed mice (for rep-resentative pictures of immunohistochemi-cal stainings, see figure 4). Dust+PBS

PBS HDM dust+PBS dust+HDM0

1

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Ainteraction p=0.001

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Figure 1: Semi quantitative analysis of expression of A) TLR2 and B) TLR4 in epithelium of PBS, HDM, dust+PBS

and dust+HDM exposed mice. Individual data points and medians are shown. “Interaction” indicates a smaller

effect of HDM exposure in dust exposed mice as compared to mice not exposed to dust. p values in the graph are

from multiple linear regression analyses.

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exposed mice had a lower expression of TLR2 and TLR4 in airway epithelium com-pared with PBS exposed mice. Dust+HDM exposure decreased the response to HDM, inducing a lower expression of both TLR2 and TLR4 in epithelium compared to PBS or HDM exposed mice (interaction).

fArm dust exposure decreAses levels of thymic stromAl lymphopoietin (tslp) in lung tissue Since downregulated TLR2/4 expression may affect TSLP production, we measured TSLP levels in lung tissue (figure 2). HDM exposed mice showed higher levels of TSLP than PBS exposed mice. Dust+PBS expo-sure induced no increase in TSLP com-pared with PBS exposure and dust+HDM exposure decreased the response to HDM (interaction), resulting in TSLP levels that were equal to TSLP levels in PBS exposed mice.

PBS HDM dust+PBS dust+HDM0.6

0.8

1.0

1.2

1.4

1.6

interaction p=0.005

p=0.001TSLP

(rel

ativ

eto

PBS)

Figure 2: Relative levels of thymic stromal lympho-

poietin (TSLP) in lung tissue of PBS, HDM, dust+PBS

and dust+HDM exposed mice. The median TSLP level

of the PBS group was set to 1. Individual data points

and medians are shown. “Interaction” indicates a

smaller effect of HDM exposure in dust exposed mice

as compared to mice not exposed to dust. p values

in the graph are from multiple linear regression ana-

lyses.

C

PBS HDM dust+PBS dust+HDM

0

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CD

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Figure 3: A) Numbers of YM1+ macrophages in lung parenchyma, B) numbers of CD68 cells beneath the epithe-

lium, C) CD68 spindle cell beneath the epithelial basement membrane and D) volume % of CD19 in infiltrates in

PBS, HDM, dust+PBS or dust+HDM exposed mice. Individual data points and medians are shown. “Interaction”

indicates a smaller effect of HDM exposure in dust exposed mice as compared to mice not exposed to dust. p

values in the graph are from multiple linear regression analyses.

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presence of the AAMΦ marker YM1. HDM exposed mice had higher numbers of YM1 expressing macrophages (figures 3A and 4) but dust+PBS and dust+HDM exposure did not result in lower numbers of AAMΦ. A striking difference between HDM exposed and dust exposed mice was that HDM ex-posed mice had more YM1 deposition in the lung parenchyma than dust+PBS and dust+HDM exposed mice (figure 4), indi-cating that AAMΦ were less active in dust exposed mice.

fArm dust exposure decreAses ym1 production by AlternAtively ActivAted alveolar macrophages (aamΦ)As we showed previously (chapter 6), HDM exposed mice had higher numbers of al-veolar (CD68 expressing) macrophages and dust+PBS and dust+HDM exposure increased the number of alveolar macro-phages even further. Since the HDM-in-duced TH2 response was downregulated in dust exposed mice and AAMΦ are known to induce TH2 reponses, we investigated the

FDCm2

CD19

HDM dust+ PBS dust+ HDMPBS

HDM dust+ PBS dust+ HDMPBS

YM1

HDM dust+ PBS dust+ HDMPBS

HDM dust+ PBS dust+ HDMPBS

TLR2

TLR4

HDM dust+ PBS dust+ HDMPBS

100 µm 100 µm 100 µm 100 µm

100 µm 100 µm 100 µm 100 µm

100 µm 100 µm 100 µm 100 µm

100 µm 100 µm 100 µm 100 µm

100 µm 100 µm 100 µm 100 µm

50 µm 50 µm 50 µm 50 µm

HDM dust+ PBS dust+ HDMPBS

CD68

Figure 4: Representative pictures of immunohistochemical staining of TLR2, TLR4, YM1 (alternatively activated

macrophages), CD68+ spindle cells, CD19 (B cells) and FDCm2 (follicular dendritic cells) in lung tissue from PBS,

HDM, dust+PBS and dust+HDM exposed mice.

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Since the number of alternatively AAMΦ was not different for HDM, dust+PBS and dust+HDM exposed mice, the decreased al-lergic airway inflammation in dust exposed mice compared with HDM exposed mice could not be attributed to lower numbers of AAMΦ. Therefore we investigated whether decreased antigen presentation in dust exposed mice contributed to decreased al-lergic airway inflammation in these mice. Both macrophages and dendritic cells can stimulate naïve T cells but a discriminative marker for these cells does not exist 18. To specifically assess the phenotype of the macrophages and/or dendritic cells in lung tissue of farm dust exposed mice an exten-sive array of markers should be investigat-ed. Since this was not the purpose of this study, we first studied the presence of the CD68+ cells in more detail. Interestingly, HDM exposed mice had more CD68+ spin-dle cells beneath the epithelial basement membrane than PBS exposed mice (figures 3B, 3C and 4), whereas the cell numbers were lower in dust+PBS and dust+HDM exposed mice compared to HDM exposed mice. These cells turned out to be YM1 negative and since CD68 is found on both macrophages and some dendritic cells, ex-pression of CD11c was investigated to see whether these cells were dendritic cells. However, no CD11c was present on these cells beneath the epithelial layer (data not shown).

fArm dust exposure increAses the num-ber of b cells And folliculAr dendritic cells in infiltrAtes

There were no B cells in PBS exposed mice whereas the volume percentage of B cells in infiltrates was higher in dust+PBS and dust+HDM exposed mice compared to HDM exposed mice (figures 3D and 4). Furthermore, dust+PBS and dust+HDM exposed mice had more follicular dendritic cells in infiltrates than HDM exposed mice (figure 4). PBS exposed mice had no fol-licular dendritic cells, since there were no infiltrates in lung tissue present at all.

fArm dust exposure increAsed igg1 production

In our previous paper (chapter 6) we showed that dust exposure prevented pro-duction of HDM-specific IgE and total IgE. Since the volume percentage of B cells in

infiltrates of dust exposed mice were high-er than in the HDM and PBS groups, we investigated which type of immunoglobulin was produced by these B cells, since it ob-viously was not IgE.HDM exposed mice displayed higher levels of IgG1 (figure 5A), IgG2a (figure 5B) and IgM (figure 5C) than PBS exposed mice. Dust+PBS exposed mice had higher lev-els of IgG1 compared with PBS and HDM exposed mice, but no difference in IgG2a and IgM compared with PBS exposed mice.

PBS HDM dust+PBS dust+HDM1

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Figure 5: Levels of A) IgG1, B) IgG2a and C) IgM

in serum of PBS, HDM, dust+PBS or dust+HDM ex-

posed mice. Individual data points and medians are

shown. “Interaction” indicates a smaller effect of

HDM exposure in dust exposed mice as compared to

mice not exposed to dust. p values in the graph are

from multiple linear regression analyses.

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Dust+HDM exposure surprisingly result-ed in a lower level of IgG1 compared to dust+PBS exposed mice.Altogether, dust exposure resulted in an increase in IgG1 levels (“dust effect”) but no difference in IgG2a and IgM levels were found.

discussion We aimed to gain more insight into the mechanisms causing downregulation of HDM-induced airway inflammation in farm dust exposed mice. Our results show that farm dust decreased the expression of TLR2 and TLR4 in airway epithelium, as-sociated with decreased TSLP levels in lung tissue. In addition, we show that farm dust exposure increased the number of B cells and follicular dendritic cells in cellular infil-trates and induced marked production of IgG1 in Balb/c mice.

TLR4 has been shown essential for HDM-induced allergic airway inflammation 15. The observed decreased expression of TLR2 and TLR4 in this study could abolish the recruitment and activation of dendritic cells that are necessary to skew the TH2 immune response. However, it was shown recently that innate immune responses of cultured epithelial cells to HDM are me-diated via CCL20 secretion, which is not TLR2 or TLR4 dependent, but relies on beta-glucan receptors 19. Therefore down-regulated expression or blocking of other pattern recognition receptors (PRRs), such as dectin-1, by farm dust exposure may have contributed to downregulation of the HDM-induced allergic airway inflammation in our study as well. These data seem to be in contrast with studies indicating that TLR2 and TLR4 signalling attenuates al-lergic airway inflammation 20-22. However, these studies were all performed in mouse models using ovalbumin (OVA) as an aller-gen, requiring sensitisation via intraperi-toneal injections of OVA and not via mu-cosal/epithelial exposure as in our study design. Therefore, different functional involvements of TLRs can be expected in these models. To investigate whether de-creased TLR expression by dust exposure could indeed abolish TH2 skewing via de-creased dendritic cell activation, we mea-sured TSLP levels in lung tissue. TSLP is an epithelium derived cytokine, involved in development of allergic airway inflamma-

tion 23-26. Hence, the lower levels of TSLP observed in farm dust exposed mice in our study may very well be involved in down-regulation of the HDM-induced allergic TH2 driven airway inflammation. The recruitment of antigen presenting cells driving the TH2 response can be decreased via lower TSLP production as well. Indeed, our data are suggestive of a decrease in antigen presenting cells beneath the epi-thelial layer in farm dust exposed mice, since we observed CD68+ spindle cells (in contrast to the round CD68+ alveolar macrophages) with long cytoplasmic ex-tensions beneath the epithelial layer in HDM exposed mice and decreased num-bers of these cells in dust exposed mice. Although it is known that this phenotype can be attributed to dendritic cells 27, we did not find expression of the epithelial cell marker CD11c on these cells. As was re-cently shown by Poole et al, organic dust exposure prevents maturation of dendritic cells, illustrated by e.g. decreased CD11c expression 28. Whether HDM and farm dust exposure indeed have differential effects on maturation of dendritic cells remains to be studied in more detail, using more den-dritic cell maturation markers. Therefore, the exact phenotype of the CD68 express-ing spindle cells remains unknown as well as whether these cells are the TH2 skewing cells in HDM exposed mice.

Another mechanism that could have con-tributed to downregulation of eosinophilic airway inflammation in farm dust exposed mice is decreased activity of alternatively activated macrophages (AAMΦ). YM1, a lectin binding protein, is a cell marker for AAMΦ 29 and is known to attract eosino-phils 30. Although we did not find differ-ences in the numbers of AAMΦ between HDM exposed mice and farm dust exposed mice, we did find that the level of YM1 ex-pression by these AAMΦ was much higher in HDM exposed mice. This indicates that farm dust exposure downregulated the ac-tivity of AAMΦ, which can have contributed to decreased TH2 responses in these mice.

We were interested in the phenotype of the cellular infiltrates in lungs of farm dust exposed mice and showed a remarkable increase in the number of B cells and fDCs in these infiltrates. This indicates that farm dust exposure induced B cell follicle forma-tion. Since TH17 cells, previously observed to be increased in farm dust exposed mice

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(chapter 6), were shown to play a role in B-cell recruitment 31 these cells could have contributed to the increased numbers of B cells in farm dust exposed mice. The marker that we used for identifying fDCs, fDCm2, is strongly expressed by fDCs in secondary follicles, but literature reports expression in primary follicles as well 32. Therefore, the presence of secondary fol-licles in farm dust exposed mice can not be indicated by this marker. Since TH17 cells were recently also proposed to drive auto-reactive germinal center formation in B cell follicles, further analysis of markers such as IL-17 receptor, Bcl-6 or peanut agglu-tinin (PNA) to indicate the presence of an (autoreactive) germinal center (and thus presence of a secondary follicle) in farm dust exposed mice, will be of interest.

Since IgE levels were lower in farm dust exposed mice than in HDM exposed mice, but the number of B cells in infiltrates was much larger in these mice, we were interested in the immunoglobulin type produced by these B cells. We show that HDM exposure increased IgG1 levels, but that the increase in IgG1 in farm dust exposed mice was far more pronounced. IgG2a and IgM were not altered by farm dust exposure. IgG is known to bind to FC receptors on macrophages 33, thereby activating them to better internalize the pathogens. Since farm dust exposed mice recruited more macrophages into the pa-renchyma, this may have contributed to increased clearance of pathogen associ-ated microbial patterns (PAMPs) present in farm dust. Another role of IgG antibodies was already suggested in the 1990s, when asthma patients with negative skin prick tests appeared to have IgG autoantibod-ies directed against endothelial proteins 34. Two more recent studies showed increased presence of IgG antibodies against epi-thelial cell antigens in non-allergic asth-ma patients compared to allergic asthma patients and healthy controls 35,36 and in severe asthma patients IgG autoantibod-ies directed against the epithelial protein alpha enolase were found 37. This indicates that an autoimmune response involving IgG antibodies, may play a role in non-al-lergic asthma. To which antigen the IgG1 antibodies in our farm dust exposed mice are directed remains to be studied, but this could prove an interesting new model to study mechanisms involved in non-allergic asthma development in farm dust exposed

individuals.

Altogether, this study suggests that farm dust exposure induces a non-eosinophilic asthma phenotype via down regulation of TLR2 and TLR4 expression in epithelium associated with lower TSLP production on the one hand and on the other hand in-duction of B cell follicle formation and pro-duction of IgG1 antibodies by B cells which may be orchestrated by the previously ob-served increased numbers of TH17 cells.

Acknowledgements This work was supported by a grant from the European Commission as part of GA-BRIEL (a multidisciplinary study to iden-tify the genetic and environmental causes of asthma in the European Community, contract No: 018996). The authors thank drs. C.A. Brandsma and M. Broekema for helpful discussions and L. Akkerman and P. Ettema for immunohistochemical stain-ings.

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fArm dust: the role of epithelium, mAcrophAges And b cell follicles

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