mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo

Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient’s individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is–in contrast to conventional viral vectors–non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis.


Introduction
Based on the principles of the hygiene hypothesis, several studies indicate that the onset of atopy and allergic asthma is less frequent in children having been exposed to an environment rich in microbes in their early childhood [1][2][3]. Increased hygiene standards in Western lifestyle however go along with a reduced contact to microbes, facilitating the development of these diseases and contributing to the rise of atopy in developed countries [4][5][6].
As primary sensors of the immune system, Toll-like receptors (TLRs) are responsible for recognizing and responding to microbes and microbial components, so-called pathogen-associated molecular patterns (PAMPs). By inducing the secretion of certain "instructive" cytokines, TLRs furthermore influence T-cell development, mainly towards a T helper cell type 1 (Th1) dominant phenotype [7]. PAMPs are involved in the pathogenesis of atopic diseases such as asthma, allergic rhinitis and allergic dermatitis. The initial triggers for these diseases are still not entirely understood. However, in the last years, multiple studies demonstrated that an imbalance of T helper cell responses plays an important role in their development [8,9]. In the case of asthma, the predominance of a Th2 pattern leads to an increased production of chemokines, as well as allergen-specific immunoglobulins, thus causing airway inflammation, eosinophilia and mucus hypersecretion in the lung [10][11][12]. The clinical presentation of atopic asthma eventually consists of wheezing, airway obstruction, breathlessness and cough, often accompanied by recurrent bronchitis or pneumonia [13,14].
The perspectives of gene therapies in the field of immunology have been of great interest in recent years. DNA-based gene therapy however implies the threat of genomic integration and immunogenicity and is furthermore often limited by low transfection efficiency. The application of nucleotide chemically modified mRNA (cmRNA) however, circumvents these threats and further ensures high stability, thus representing a promising therapeutic tool [15][16][17][18][19]. Previous work by our group and others has shown that delivery of cmRNA leads to therapeutic levels of protein expression as a result of high gene transfer efficiency, higher stability and/or low immunogenicity, and hence, can even be utilized for live-saving genome editing in vivo [17,19,20].
We found that polymorphisms in TLR1, 6 and 10, all capable of forming heterodimers with TLR2, have shown protective effects on atopic asthma in humans [21]. These effects were further associated with an increased expression and elevated peripheral blood mononuclear cell secretion of Th1 cytokines. Recent studies suggest a protective role of TLR6 activation in asthma via the regulation of cytokine expression by dendritic cells [22].
Here, based on the data of asthma-protecting TLR-haplotypes, we investigated the intratracheal application of combinations of chemically modified Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite (HDM)-induced mouse model of asthma. We further analyzed how this treatment in vivo differentially modulates neutrophilic and eosinophilic airway inflammation and lung function.

Animal experiments
Female BALB/c mice were purchased from Charles River Laboratories at an age of six to eight weeks. Mice were kept under specific pathogen-free conditions and maintained on a 12-h light-dark cycle. Food and water were provided ad libitum. All animal experiments were approved by the ethics committee of the regional board of Tübingen and carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals at the University of Tuebingen, the German Law for the Protection of Animals and FELASA regulations. All efforts were made to minimize suffering of the animals. Intratracheal procedures were carried out using a high-pressure spraying device (PennCentury) under antagonizable anesthesia with a mixture of medetomidine (0.5 mg/kg), midazolam (5 mg/kg) and fentanyl (50 μg/kg). After treatment antidot (atipamezol (50 μg/kg), flumazenil (10 μg/kg) and naloxon (24 μg/kg)) was injected s.c. Per time point, mice received 100 μl DEPC-water containing either 20 μg of the respective Tlr mRNA or water as control. HDM (Greer Laboratories) was administered as 100 μg extract dissolved in 100 μl PBS. At experimental endpoints, animals were euthanized using 120 mg/kg Na-Pentobarbital.

Cell preparations
Lungs were lavaged with 1 ml PBS in order to obtain BALF. Total BAL cells were subsequently centrifuged and analyzed. Differential cell counts were performed by counting at least 100 cells (macrophages, monocytes, neutrophils, eosinophils, basophils and lymphocytes) from different fields of view on cellspin preparations (CellspinII, 6°C, 75 g, Ramp 4, Break 6; Tharmac). Lungs were harvested and incubated for 1 hour at 37°C in a digestion solution containing 1 mg/ml collagenase type 1 (Life Technologies), 1 mg/ml Dispase (Corning) and 500 U DNase (EPI-CENTRE Biotechnologies). Thereafter, digested lungs were passed through a 70 μm cellstrainer in order to receive a single-cell suspension. Erythrocytes were lysed using ACK-Lysing Buffer (Life Technologies) and cells were counted and subsequently subjected to flow cytometry (FACS) analysis. For

Airway resistance
At the predetermined endpoint of the study, airway resistance in response to methacholine (Sigma-Aldrich) was determined using the ex vivo model of the IPL as previously described [19,20]. In short, in situ mouse lungs were placed in a thorax chamber and mice were ventilated via a tracheal cannula. Ventilation was set to 90 breaths/minute with negative pressure ventilation between -2.8 cm H 2 O and -8.5 cm H 2 O. To prevent atelectasis, a hyperinflation was triggered every 5 min (-25 cm H 2 O). Perfusion of lungs was done with a 4% hydroxyethyl starch (HES 200/0.5, Serumwerk Bernburg) containing perfusion buffer through the pulmonary artery (1 ml/min). Lung function parameters were recorded automatically and resistance measured by HSE-HA PulmodynW Software (Harvard Apparatus). After a 20-minute equilibration period, lungs were perfused with increasing concentrations of MCh (0.1 μM, 1 μM, 10 μM, and 100 μM) for 10 minutes each, separated by a washout period (10 min) with buffer. For graphical and statistical analysis, the mean resistance values were calculated from the last ten time stamps of each 5-min period.

Histopathology
Whole lung tissue sections were fixed in Histofix (4.5%, Carl Roth) overnight and embedded in paraffin. Slices (4 μm) were stained with either H&E or PAS and examined using a Zeiss Axio Imager.M2 with the AxioCam MRc camera. Tissue inflammation and infiltration was evaluated on H&E stained sections. PAS-positive goblet cells were quantified in percent of counted cells which have been determined by visible nuclei.

Statistics
Statistical significance of differences was defined as a P<0.05 and denoted with asterisks: Ã 0.05, ÃÃ 0.01 and ÃÃÃ 0.001. All calculations were performed using GraphPad Prism 6.0 (GraphPad Software) and SPSS Statistics Version 22 (IBM). If not stated otherwise, data were statistically analyzed using Kruskal-Wallis one-way-analyses and Mann-Whitney U tests.

Results and Discussion
Mice received combinations of Tlr mRNA (Table 1) at four determined time points (-17, -14, -10 and -7 days) before sensitization and challenge with HDM (Fig 1A). At day 15, the predetermined endpoint of the study, mice were sacrificed and analyzed.
Bronchial alveolar lavage fluid (BALF) cells were obtained at the time of sacrifice and analyzed via differential cell count ( Fig 1B). Here, delivery of Tlr1/2 mRNA led to decreased levels of neutrophils and eosinophils in BALF, when compared to untreated controls. Treatment with Tlr2/6 mRNA resulted in higher amounts of eosinophils but still reduced neutrophilic inflammation. Levels of neutrophils were increased after the administration of Tlr2 mRNA, eosinophils and lymphocytes were slightly diminished. Representative micrographs shown in Fig 1C additionally illustrate the decline of inflammatory cells after administration of Tlr1/2 mRNA when compared to the HDM group.
In order to investigate the local effect of Tlr mRNA application on immune cells in lung tissue, we isolated and stained lung cells and subjected them to FACS analysis (Fig 1D). Similar to observed results in the BALF, delivery of Tlr1/2 mRNA was able to reduce the number of eosinophils in lung tissue. Tlr2 mRNA led to a rise of eosinophils and furthermore markedly increased levels of neutrophils. Also treatment with Tlr2/6 mRNA resulted in higher levels of neutrophils and did not reduce eosinophilic inflammation in lung tissue.
In lung sections, either stained with H&E or PAS, we observed markedly reduced peribronchial, perivascular and interstitial tissue inflammation in lungs of mice treated with Tlr1/2 mRNA (Fig 2A). Delivery of Tlr1/2 mRNA was furthermore associated with a significant reduction of goblet cells in airways (P = 0.007) (Fig 2B). Administration of Tlr2 mRNA did not  dampen lung inflammation (Fig 2A) and resulted in a rather higher degree of goblet cell metaplasia when compared to untreated HDM controls (Fig 2B). Lung tissue inflammation was slightly reduced after treatment with Tlr2/6 mRNA, whereas no differences in goblet cell metaplasia could be detected. Next, we wanted to determine whether the administration of combinations of chemically modified Tlr1, 2 and 6 mRNA modulates airway hyperresponsiveness of mice in HDMinduced asthma. We therefore measured lung function in terms of airway resistance by using the ex vivo model of the isolated, perfused and ventilated lung (IPL).
We observed considerably reduced airway resistance values following ex vivo methacholine (MCh) challenge after delivery of Tlr1/2 mRNA prior to HDM administration (Fig 2C). Likewise, application of Tlr2/6 mRNA was associated with diminished airway resistance, indicating decreased airway hyperresponsiveness in these two groups. However, delivery of Tlr2 mRNA led to slightly higher resistance values than in untreated asthmatic controls and thus to a decline in lung function.
In conclusion, we observed that intratracheal administration of Tlr1/2 and Tlr2/6 mRNA resulted in markedly reduced lung inflammation. Tlr1/2 mRNA application led to a concomitant improved lung function in vivo. Administration of Tlr2 mRNA alone showed no improvement compared to untreated HDM controls.
As mentioned before, previous studies presented protective effects of TLR1 and 6 on atopic asthma in humans [21,31]. In line with these findings, we observed that treatment with the combination of Tlr1/2 -and to some extent Tlr2/6 mRNA-tended to result in a better asthma outcome in vivo. Percentages of neutrophils and eosinophils in BALF were reduced after Tlr1/2 administration when compared to the HDM control group, also absolute numbers of eosinophils in lung tissue were diminished. Furthermore, these mice showed notably improved lung function and significantly reduced pulmonary mucus production.
However, these findings did not hold true for treatment with Tlr2 mRNA alone. In contrast, Tlr2 mRNA treatment promotes a rather proinflammatory phenotype regarding not only BALF and lung tissue but also lung function and histological analyses. These observations are in line with reports describing a crucial role of TLR2 being overexpressed in fatal asthma patients [32] and in patients suffering from persistent allergic rhinitis [33]. In a study investigating an ovalbumin-induced mouse model of asthma, the allergic response appeared to be largely TLR2 dependent, with significantly reduced allergic immune responses in TLR2-deficient mice [34]. The specific interaction of HDM components with TLR2, promoting a Th2 biased allergic immune response and close cross-talk between receptor pathways might serve as another explanation for this observation [35].

Conclusions
Allergic asthma is a major burden worldwide and although several therapeutic options are available to treat asthma symptoms, especially cases of severe asthma are still difficult to control [36][37][38]. To target individual patient's needs and provide tailored treatment, clinical approaches in the field of immunotherapy to target TLRs and Th responses are already subject  [39]. Due to the rather small number of mice, the present data should be interpreted in the context of the experimental design and considered as a pilot-study for future research. In this regard, analysis of TLR1/2 and/or TLR2/6 overexpressing mice would be of special interest. Despite some limitations of the study, our data point to a potentially protective effect of Tlr1/2 mRNA treatment on HDM induced asthma. New insights into the role of TLRs in atopic asthma combined with novel therapeutic tools, such as cmRNA transcripts, can be considered promising targets of future research in the field of asthma management and prevention.
Supporting Information S1