BRONCHIOLAR EPITHELIUM PARTICIPATION IN THE NEONATAL PREVETION OF ASTHMA
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1
Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Instituto de Investigaciones en Ciencias de la Salud (INICSA)-CONICET, Argentina
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2
Sanatorio Allende, Neumonología, Argentina
INTRODUCTION:
Although the susceptibly for allergic disease has a hereditary element, the current worldwide raise in asthma prevalence have evidenced that environmental changes are also implicated. A possible explanation for this phenomenon has been suggested by the so-called hygiene hypothesis, based on epidemiological studies. This hypothesis states that the urban hygienic conditions skip the early-life exposure to microbes hence they are clue to promote innate immune activation by signaling through the Toll-like receptors (TLRs) and thus preventing the allergic response. Even though the way this microbial stimuli exert such prevention is still not well know, the use of murine models have highlighted the possible compensatory mechanisms involved.
Nowadays, a growing body of genetic and clinical data highlights the need to consider the role of structural components of the airway in the onset and development of asthma. In particular the epithelium is central, because this is the site where an inflammatory or non-inflammatory decision must first be made to inhaled allergen. In these sense, at bronchiolar level, the epithelial Club cells (CC) are key contributors for local homeostasis.
Under normal conditions these cells detoxify xenobiotics as well as oxidant gasses, participate in mucociliary clearance of environmental agents and through their pluripotencial capacity, they are involved in the epithelial maintenance and repair. In addition, these cells modulate the local inflammation by secreting the anti-inflammatory Club cell secretory protein (CCSP) and participate in the innate immune response through the secretion of the collectins surfactant protein (SP) A and D as well as several cytokines and chemokines. Among these mediators, it has been reported that both, CCSP and SP-D, exhibited Th2- immunomodulatory effect.-Beside these lung protective functions, CC also have been linked to the allergic remodeling process of asthma, since they are the principal cells to produce eotaxin and undergo mucus metaplasia in response to Th2 inflammation.
Previous results of our laboratory indicate that this mucous transdifferentiation induced the diminution of the normal SP-D and CCSP content, while the intranasal stimulus with Lipopolysaccharide (LPS) increase the Club cell expression of these proteins as well as the receptor TLR4.
OBJECTIVE:
The aim of this study was to modify the microbial environment to which lung epithelium of newborn Balb/c mice are exposed and study the possible influence on the subsequent development of airway allergic inflammation to Ovolabumin (OVA).
MATERIAL AND METHODS
Offspring Balb/c mice were intranasal (in.)pre- exposed to 5μl of PBS or E. Coli Lipopolysaccharide-LPS (200mg/ml), 3 times per week , between days 3 to 13 after birth. At the age of 4 and 6 weeks, female mice were systemically sensitized by means of 100μl intraperitoneal injection of OVA/Alumm (1mg/1mg/ml). Then, mice were assigned into groups (n=10) and in. challenged on 10 consecutive days with OVA (1mg/ml) (LPSn/OVA and PBSn/OVA group) or vehicle (LPSn and PBSn group). Twenty four hours later animals were sacrificed, bronchoalveolar lavages (BAL) were performed and lung tissues were obtained. After centrifugation BAL supernatant were reserved at -20°C for ELISA assay while pellet were used for flow cytometry (FC) analysis and differential cell count in cytospin slide with May Grünwald-Giemsa staining. The right lungs were processed for electron and light microscopy analysis as well as immunohistochemistry (IC) and Alcian Blue (AB)-PAS staining.
Moreover, to further investigate the changes primed by LPS-neonatal instillation in the bronchiolar epithelium, a group of animals (n=6) (equally exposed to PBS or LPS) were sacrificed at 20 days of live and epithelial-bronchiolar samples were obtained (20/animal) by lasser dissection microscopy. RNA extractions followed by qRT-PCR were conducted in these samples.
RESULTS
Ultrastructural analysis demonstrated in LPSn/OVA the preservation in the normal phenotype of CC as well as a meaningful reduction (p<0,001) in AB-PAS positive cell in bronchiolar epithelium compared to PBSn/OVA, implicating a meaningful prevention of the CC mucous metaplasia.
Bronchiolar CC of LPSn and LPSn/OVA mice also demonstrated an increased expression of host defense molecules like SP-D and CCSP and TLR-4 by IC. Accordingly, although the innate immune mediators CxCL1, CxCL10 and TNFα were expressed in bronchiolar epithelium, the qRT-PCR showed that only TLR4 levels were increased by the neonatal LPS stimulus (p<0,05).
The BAL analysis showed a reduction in the allergic inflammatory microenvironment in LPSn/OVA, since the number of eosinophils (p<0,01 vs PBSn/OVA) were reducer and normal levels of the Th2 related cytokines: IL-4 and TSLP were detected by ELISA. In addition, LPSn and LPSn/OVA mice exhibited elevated levels of TNFα (p<0,05 vs PBSn) and IL-12 (p<0,01 vs PBSn). However, only in LPSn group the Th1 cytokine: IFNγ increases (p<0,001vs PBSn), suggesting that the allergen stimulus in LPSnOVA group provoked another Thelper cell subset. Accordingly, in LPSn/OVA group the percentage of CD4+FOXP3+ Treg cells present in the BAL were increased (p<0,01 vs PBSn), although the IL-10 levels was not detectable in the supernatant of experimental group.
CONCLUSION
All together, these results are indicative of the long-term preservation of innate defenses promoted in airway epithelium that protected from a Th2-biased and contributed to a more balanced immune response in front to allergens.
Acknowledgements
The authors are thankful to Prof. Virginia Rivero and Martin Rumbo as well as their staff member.
This work was supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) [Nº 11220100100280] and the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [PICT 2012-0654].
Keywords:
Asthma,
Bronchiolar Epithelium,
TLR ligand,
Host defence peptides,
neonatal immunology
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Allergy
Citation:
García
LN,
SCALERANDI
MV,
Uribe Echevarría
EM,
Leimgruber
C,
Quintar
AA and
Maldonado
CA
(2015). BRONCHIOLAR EPITHELIUM PARTICIPATION IN THE NEONATAL PREVETION OF ASTHMA.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00257
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Received:
29 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
MD. Luciana N García, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Instituto de Investigaciones en Ciencias de la Salud (INICSA)-CONICET, Córdoba, Argentina, lgarcia@cmefcm.uncor.edu