Neonatal endotoxin stimulation was associated with long- term innate immune markers and an anti-allergic response in bronchiolar epithelium in spite of allergen challenge

Asthma is a heterogeneous disease underlying different medical processes, being the allergic asthma, with an early-onset in childhood, the most common type. In this phenotype, the continuous exposure to allergens produces a Th2-driven airway remodeling process that leads to symptoms and pathophysiological changes in asthma. Strategies as the avoidance of aeroallergen exposure in early life have been tested to prevent asthma, without a clear success. Alongside, several mouse models of aeroallergen challenge have dissected potential homeostatic responses by which environmental microbial stimulation reduces the subsequent allergic inflammation in the offspring. This suggests the onset of underlying preventive mechanisms in the beginning of asthma that have not been fully recognized. In this study, we aimed to evaluate if neonatal LPS-induced stimulus in epithelial host defenses could contribute to the prevent asthma in adult Balb/c mice. For this purpose, we studied the response of bronchiolar club cells (CC) that are situated in the crossroads of the host defense and allergic inflammation, and express specific pro and antiallergic proteins. LPS stimulus in the neonatal life intensified the production of TLR-4, TNFα, and natural anti-allergic products (CCSP and SPD), changes that contributed to prevent asthma triggering in adulthood. At epithelial level, CC skipped the mucous metaplasia, declining the overproduction of mucin via the EGFR pathway and the mice expressed normal breathing patterns in front of OVA challenge. Furthermore, the overexpression of TSLP, an epithelial pro-Th2 cytokine was blunted and normal TSLP and IL-4 levels were found in bronchoalveolar lavage (BAL). Complementing this shift, we also detected lower eosinophilia in BAL while an increase in phagocytes as well as in regulatory cells (CD4+CD25+FOXP3+ and CD4+IL-10+) was seen, whit an elevation in IL-12 and TNFα secretion. Summarizing, our study pointed to stable asthma-preventive effects promoted by neonatal LPS-stimulation; the main finding was the increase of several anti-Th2 specific proteins at epithelial level, together with an important diminution of pro-Th2 TSLP, conditions that promoted changes in the local immune response with Treg. We thus evidenced several anti-allergic dynamic mechanisms overlying in the epithelium that could be favored in an adequate epidemiological environment


INTRODUCTION
Asthma is a heterogeneous disease with diverse underlying processes and many 57 clinical expressions. The most common phenotype is the allergic asthma that has an 58 early-onset in childhood. This phenotype is associated with a family history of allergic 59 diseases, and is characterized by chronic airways inflammation, with activated mast 60 cells, increased numbers of eosinophils, T cells, natural killer T cells, and CD4+ T 61 helper (Th) 2 cells that release IL-4, IL-13, and IL-5. Additionally, IgE-secreting B cells 62 are induced during the asthma process (1, 2). In this phenotype, the continuous exposure 63 to allergens produces several consequences in the structure and function of the airways, 64 with the establishment of a remodeling process that includes mucus hypersecretion,   Additionally, there is accumulative evidence about AECs intrinsic alterations in 98 childhood asthma that render airways more vulnerable to airborne allergens and 99 predispose them to Th2-responses (31-34). These data indicate that AECs are essential 100 controller of the immune response to allergens and may be an early player in order to 101 bias a Th2 response in the immature immunity system. Therefore, AECs play a 102 particular role since they are situated at the crossroad of the innate host defense and 103 allergic inflammation.

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The potential of CC to respond to Th1 inflammatory stimulus, activating protective 119 mechanisms, has often been applied in studies to evidence if this protective role of 120 epithelium prevents the development of Th2 inflammation. In a previous study we 6 121 reported that LPS pre-exposition to the allergen sensitization partially avoids mucous 122 metaplasia of CC. In consequence, the loss of anti-allergic products in CC and alveolar 123 adult mice macrophages were prevented. We observed a reduction of eosinophil influx, 124 Interleukin-4 levels and airway hyperreactivity, while the T-helper type 1 related 125 cytokines IL-12 and Interferon-g were enhanced (55). Considering early life as a better 126 window of opportunity for triggering an appropriate maturation of innate immunity. In 127 this study, we aimed to evaluate if LPS-stimulation during the neonatal lapse provides 128 better asthma-preventive effects to preserve adult AECs from the Th2-driven      Airway challenge: Ten days later, neonatally (n) LPS-treated mice as well as PBS-154 exposed were divided into 2 groups. Whereas LPSn/OVA and PBSn/OVA mice were 155 challenged daily (on 10 consecutive days) by an intranasal application of 50μl of 1% 156 OVA, LPSn and PBSn mice were submitted to intranasal application of saline ( Fig 1a).
Then, after 24h, mice were sacrificed and processed according to the specific methods 158 outlined below.          LPSn/OVA mice compared to the PBSn/OVA group (Fig.1b). In addition, the number 316 of macrophages remained unchanged while neutrophils increased significantly (Fig 1a).
317 Surprisingly, IL-4 and TSLP, both associated to Th2 inflammation, exhibited normal 318 levels in BAL of LPS pre-treated mice in spite of the allergen-challenge, while they 319 were significant higher in PBSn/OVA group (Fig. 1c and 1d respectively). As expected, 320 in the LPSn group neither BAL cell count nor IL-4 content were different from controls 321 (Fig 1b and c); TSLP was remarkable reduced to non-detectable levels (Fig. 1d).

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To test whether the inflammatory parameters were accompanied by changes in 323 the degree of respiratory distress, a clinical scoring system was carried out (See 324 Supplementary material). While most of the neonatal PBS-exposed mice displayed 325 higher signs of respiratory distress after OVA challenge, the breathing pattern of 326 LPSn/OVA mice was not different from control mice (Fig 1e). cytokines as TNFα and IL-12 in both LPSn and LPSn/OVA mice groups ( Fig. 2c and   334 2b, respectively). It was noteworthy that LPS induced high levels of IFNγ, a 335 prototypical Th1 cytokine, in the LPSn group but not in LPSn/OVA animals (Fig. 2a).
By contrast, the latter exhibited the higher influx of CD25+/FOXP3+ Treg cells (48,16 337 % ± 10,2 LPSn/OVA vs 19.65 % ± 4,22 PBSn/OVA) (Fig.2d). Furthermore, LPSn/OVA) compared to control. (Fig. 2e).   PBSn/OVA mice (pEGFR panel in Fig. 3a). Whereas in LPSn/OVA mice, both, pEGFR 364 overexpression (pEGFR panel in Fig. 3a) and mucous metaplasia (Fig. 3b), were largely 365 reduced by the neonatal endotoxin-treatment.  Fig 3a). These results were corroborated by the quantitative PCR 385 analysis (Fig. 3c) 390 Garcia 2014). For this reason, we studied CC morphological profile in all groups by 391 electron microscopy (Fig. 4). At this level, we corroborated the preservation of the 392 typical cellular profile in PBSn mice, characterized by the presence of a dome-shape 393 cupola, numerous polymorphic mitochondria in the cytoplasm, along with scarce 394 spherical electron-dense secretory granules under the plasma membrane (Fig. 4a). These 395 parameters could also be seen in LPSn/OVA mice, which differed only by an increase 396 on the number of normal electron-dense granules as well as a mayor development of 397 RER (Fig. 4d). Meanwhile, PBSn/OVA animals displayed characteristic mucous cell 398 metaplasia featured as a hypertrophied cytoplasm filled up with numerous large 399 electron-lucent secretory granules, slim mitochondria and abundant RER (Fig. 4b). In 400 control mice, only exposed to LPS, CC also developed an increased number of electro-401 dense granules, as was shown by LPSn/OVA animals (Fig. 4c). In this group, the 402 evident diminution of their CC cupola is probably due to the repeated LPS instillation   (Fig 5a). These changes in protein expression of CCSP and SP-D were also 420 verified by immunoblottting ( Fig. 5b and 5c). However, the neonatal LPS-instillation  Regarding the microbial recognition and cytokine response, both the toll like 427 receptor 4 (TLR4) and TNFα, increased their expression in CC as well as in lung tissue 428 of both LPS neonatal stimulated groups (Fig 5d and 5e, respectively). These suggest a 429 specific LPS-response in bronchiolar epithelium that induced a persistent elevation of 430 these defense molecules and seemed to be preserved in spite of allergen stimulus.  In the present work, we reported that neonatal LPS-treatment triggers anti-447 allergic secretory products of the local airway epithelium that persist in adulthood.

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Among these products, we demonstrated, the increase of CCSP and SP-D content, As expected, an increase in TNFα and IL-12 was observed in both groups exposed 480 to LPS; nevertheless, a robust Th1 response was only seen in LPS exposure animals as

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Although the correlation of Treg and host defense molecules described is similar to our 513 results, our finding pointed to the epithelium involvement in this persistence immune 514 response.

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As was descripted before, several experimental and clinical studies established the 516 correlation between LPS pre-exposure and asthma phenotype abrogation; our study 517 attempted to dissect the changes of a pro-allergic cytokine secreted by the epithelium 518 such as TSLP in this context. In this sense, our results demonstrated that LPS neonatal 519 exposition correlated with the abrogation of TSLP expression in epithelium and in BAL.  The main contribution of our study is highlighting the involvement of