Abstract

The increased interest in the process of autophagy in various physiological and pathological conditions is the focus of this study. Therefore, the aim was to assess the involvement of lymphocyte autophagy in the pathogenesis of bronchial asthma. Forty-five patients with mild persistent asthma, 45 patients with severe persistent asthma, and 45 normal healthy controls were involved in this study. Autophagy was evaluated based on the expression of microtubule-associated protein light chain 3 (LC3) by western blot, Fluorescence microscopy, and flow cytometry. Transmission Electron Microscopy was used to detect autophagosome. The results showed that autophagy was activated in T-cells of patients with mild and severe asthma compared to the normal healthy control. Stress conditions induced autophagy in T-cells of asthmatic patients with mild and severe form, but not in the control group. Dexamethasone treatment of T-cells stimulated apoptosis in mild asthma patients and in the control group but in the group with severe asthma, dexamethasone induced autophagy. Thus, autophagy could play an important role in the pathogenesis of asthma especially of severe asthma, and may contribute to the survival and the activation of T-lymphocyte in patients with severe asthma.

1. Introduction

During the studies of apoptosis, it became clear that cell death path is not unique ¹. Another candidate is autophagy ², a fundamentally physiological process that plays an important role in the turnover of cellular proteins and other macromolecules ³. At cellular level, autophagy is mainly important for cell protection and cell survival. Nevertheless, autophagic cell death occurs and which is considered as programmed cell death type II ⁴.

Studies showing the existence of autophagy cellular death bring to light either the occurrence of an autophagic phenomenon using classical markers and or the subsequent cell death without having ineludibly the cause and effect relationships ⁵. It could also occur during pathologies such as cardiovascular diseases, cancer, metabolic disorders, neuro-degenerative diseases ^{6, 7}. For example, autophagy has been described in Alzheimer and Parkinson diseases ⁸. Otherwise, some studies suggested that autophagy may be involved in asthma ⁹.

Asthma is a “disease of reversible airflow obstruction characterised clinically by wheezing, shortness of breath, and coughing” ¹⁰. Its medical care involves inhaled corticosteroid ^{11, 12, 13}. But this treatment encounters problems of loss of effectiveness with about 10% of asthmatics ¹⁴. Some patients face persistent symptoms, obstruction of air flow and chronic inflammation with exacerbations ¹⁵. They are called severe asthmatics ^{12, 14, 15, 16}.

One of main characteristics of asthma is airway inflammation. Multicellular inflammatory involves eosinophils, neutrophils, T helper cells and epithelial cells and contribute to asthma pathogenesis ¹⁷. Ban et al. ¹⁸ studied the association between asthma severity and autophagy of eosinophils and airway epithelial cells. Other studies showed the association between prolongation of allergic inflammation and the increased survival of T-lymphocytes and the loss of their ability to undergo apoptosis ¹⁹. Furthermore, the role of autophagy of T-lymphocytes in the pathogenesis of asthma is little known. The aim of the present study was to investigate the morphological and biochemical features of autophagy in peripheral blood T-cells (PBTC) from patients with mild and severe bronchial Asthma.

2. Material and Methods

The study included a group of 45 male patients with mild persistent asthma (MA) (mean age 42 ± 5 years), a group of 45 male patients with severe persistent asthma (SA) (mean age 42 ± 5 years) and a group of 45 normal male healthy controls (NCs) (mean age 42 ± 5 years). All patients and controls had no other chronic diseases. All subjects were non-smokers, did not receive corticosteroids in the 2 weeks prior to recruitment for the study and were selected after informed formal consent had been obtained. All asthmatics had a history of intermittent chest tightness, wheezing or shortness of breath for at least 5 years prior to study participation consistent with the diagnosis of asthma according to Global Initiative for Asthma. None of the individuals had any respiratory infections in the month previous to their inclusion in the study. No other clinically relevant diseases were reported. The work was performed in accordance with the rules of the Ethics Committee in the laboratory of Clinical Immunology and Allergy of RKB and with the regulations of the Ministry of Health of the Russian Federation in compliance with the Helsinki Declaration ²⁰. Venous blood from the subjects was drawn into a BD Vacutainer® containing K3 Ethylenediaminetetraacetiq acid (EDTA) (Greiner Bio-one, Austria) and processed within 2 h after collection.

T lymphocytes were isolated from peripheral blood by centrifuging the blood through a Ficoll density gradient (ρ=1,077g cm^-1) ²¹. Negative isolation by superparamagnetic CD3 beads (Dynabeads CD3, DYNAL, Invitrogen) was used for the getting a pure population of T lymphocytes according to the manufacturer’s instructions. The viability of T lymphocytes was determined by the trypan blue exclusion method ²² and observed under an inverted microscope.

Peripheral Blood T cells (PBTC) (2x10⁶ cells per milliliter) were cultivated in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), antibiotics (5,000 units Penicillin per ml / 5,000 µg Streptomycin per ml) (Invitrogen) and 1% L-Glutamine (Invitrogen) during 3 days at 37°C in a humidified air atmosphere with 5% CO₂. As there is currently no information on the effect of dexamethasone on the process of autophagy, we evaluated the therapeutic effect of dexamethasone on the process of autophagy. Cells were treated with dexamethasone (10^-4M in final concentrations) for up to 3 days. Pepstatin A (15 μm) (E + P) were used to block the degradation of autophagolysosomes by inhibiting lysosomal proteases.

PBTC were fixed at room temperature in a 0,1 M phosphate-buffered solution (pH 7.2) containing 2.5% glutaraldehyde (Serva) for 1,5 h and then post-fixed in 1% osmium tetroxide (Serva) for 2 h. Samples were then dehydrated in ascending concentration of ethanol, acetone and propylene oxide and embedded in Epon 812 (Serva). Samples were polymerized for three days in the incubator at 37°, 45° and 60°C, respectively. The ultrathin sections were prepared using LKB III ultramicrotome (Sweden). Then, it was stained with 2% uranyl acetate in 50% methanol and lead citrate and then examined by a transmission electron microscope Hitachi-125 (Japan). The specimens were observed by TEM to visualize the autophagosomes in the cell cytoplasm.

PBTC were lysed after culturing in buffer containing 50mM Tris pH 7.5, 1% Triton X-100, 0.1% (w/v) sodium dodecyl sulfate, 150mM NaCl, 200mM DTT for 5 min at 95ºC on heat block. Then the samples were put directly on ice, cooled down, spined, marked and stored at . The samples were thawed slowly on ice (30 min) then sonicated (UP100H, Hielscher) to break the genomic DNA. Centrifugation was performed at 10 000 rpm for 2 min the samples after sonication. 5 μl of 1 % added to each sample before loading. Cell lysates containing ~50μg of protein were separated by 4-20% gradient SDS-PAGE and transferred in transfer buffer (25mM Tris, 192mM glycine and 20% methanol, pH 8.3) to nitrocellulose membrane (Invitrogen, Molecular Probes) for 1 hr at 350 volts. LC3 rabbit mAb and β-Tubulin rabbit mAb (Cell Signaling Technology) was used. As the secondary antibody, anti-rabbit HRP-conjugate (Cell Signaling Technology) was used. Bands on immunoblots were visualized by adding Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Molecular Probes).

Flow cytometry was performed to detect and quantify autophagic vacuole. Cells were stained with LC3B rabbit monoclonal antibody (Invitrogen, Molecular Probes) and secondary goat anti-rabbit IgG antibody (Alexa Fluor 488, Invitrogen, and Molecular Probes) and run on a flow cytometry FACSCalibur (Becton Dickinson). Detection of fluorescence was carried out using the Cell Quest software. Each sample was collected and 10000 events were analyzed.

Imaging of autophagosome was performed after 3 days cultivation, using FITC-labeled antibodies against the protein LC3B (LC3B Antibody Kit for Autophagy, Invitrogen, and Molecular Probes) for fluorescence microscope Carl Zeiss AxioScope A1 (equipped with a video camera AxioCam MRc5).

Statistical analysis was performed using STATISTICA 6.0. In analyzing the data, conventional methods of parametric and non-parametric statistics were used. Differences were considered significant at p<0.05.

3. Results and Discussions

Long term cultivation of cells was accompanied by the depletion of nutrients in the medium that was used as stimulating factor for the induction of autophagy. That's why the PBTC from asthmatic patients (MA and SA) and from NCs were cultivated in complete medium for 72 hours. Detection of autophagosomes by TEM was a hallmark of autophagy.

• Cell changes with T lymphocytes of Healthy donors

After 3 days of cultivation, most of T lymphocytes of healthy donors presented a morphology corresponding to apoptotic changes at an early stage, such as the loss of cell membrane microvilli, decrease of the nucleus volume and chromatin condensation at the periphery (Figure 1, A). Autophagosomes were not identified. The culture of T cells with dexamethasone led to stimulation of the apoptotic process. We detected the chromatin condensation, blebbing and deep invagination of the nuclear and plasma membranes (a sign of the final stage of apoptosis) (Figure 1, B). The activation of autophagy (the presence of autophagosomes in the cells) was not detected.

• Cell changes with T lymphocytes of Patients with MA

Microphotographs of T lymphocytes from patients with MA showed that most of cells kept morphology of typical proliferating cells (Figure 2, A). Representative images illustrate two vacuoles with degraded intracellular components for 72 hours of cultivation without dexamethasone. These cells contain autophagosomes with differentiated cellular organelles (Figure 2 A, B). In addition, we found fragments of dead cells containing autophagosomes (Figure 2, C). Thus, in T-cells of patients with MA, initiation of autophagy promotes cell death when apoptotic activity in cells is not sufficient. Our study on this group of patients also shows that dexamethasone stimulates apoptosis and inhibits autophagy in T lymphocytes (Figure 2, D).

• Cell changes with T lymphocytes of Patients with SA

In T-cells of patients with SA, stress conditions stimulated apoptotic changes such as сhromatin condensation, cytoplasmic vacuolization and the reduction of microvilli on the cell surface (Figure 3, A). Moreover, at the same time the presence of a significant amount of autophagosomes was observed (Figure 3, A, B). It appears that, in this case, autophagy is necessary for induction of apoptosis. In the group of patients with SA, the culture of PBTC with dexamethasone contributed to intense activation of autophagy: a marked increase in the number and size of autophagosomes in the cells (Figure 3, C, and D). Apoptotic manifestations are negligible.

Immunoblots were performed for LC3 in T-cells. LC3 undergoes regulated modifications during the induction of autophagy, delivering a faster-migration processed band (termed LC3-II) to autophagosomes. The conversion of 18 kD LC3 I to the 16 kD LC3 II correlates with autophagy induction ^{23, 24}. Therefore, LC3 immunoblotting was used to test whether dexamethasone induces autophagy in T cells of different studied groups.

• Biochemical features of autophagy in the group of Healthy donors

Autophagic marker protein was present in the total (LC3-I) form which was found in the cytoplasm and was not associated with the membrane of autophagosome. Dexamethasone treatment for 72 hours did not appear to increase the levels of LC3 II (Figure 4, hole 2, 3). Fluorescence microscopy showed that in T lymphocytes of NCs, LC3-II protein expression was absent (Figure 5, A).

• Biochemical features of autophagy in the group of MA

In the group with MA, the LC3 protein was represented only by form I (Figure 4, well 4) and this was not confirmed by the electronic microscopy study (Figure 2, A, B). It can be assumed that the content of LC3-II protein in the cells was not enough to influence its detection. Another cause of the loss of LC3-II form was a rapid degradation of LC3 II by lysosomal proteases following the fusion of autophagosomes with lysosomes, making interpretation of LC3 immunoblotting results problematic. Dexamethasone treatment showed that the glucocorticoid was not responsible for the formation of LC3-II protein in the group with MA (Figure 4, hole 5). The LC3 immunoblotting results confirmed the electron-microscopic study of T lymphocytes of these patients which showed that dexamethasone promoted intense activation of apoptosis, but not autophagy (Figure 2, D). Autophagosomes were present in T cells of patients with MA (Figure 5, B). The expression of LC3 protein in the cells from patients with MA was decreased by dexamethasone-treatment (Figure 6, B).

• Biochemical features of autophagy in the group of SA

In cell lysates of patients with SA the LC3 protein was presented in two forms: LC3-I and LC3-II (Figure 4, hole 6). Moreover, dexamethasone treatment arose from LC3-I to LC3-II conversion (Figure 4, hole 7). Autophagosomes were present in T cells of SA (Figure 5, C). The amount and intensity of LC3-II protein expression was higher in patients with SA. There was a significant increase of LC3 protein expression in T-cells from patients with severe asthma following dexamethasone treatment for 72 hours, backing up the conclusion that dexamethasone induces autophagy in T-cells of patients with asthma (Figure 6, C). Based on these findings, we concluded that it is not only stressing conditions that induce autophagy in PBTC of asthmatic patients but cells could also undergo autophagy by Dex treatment as well. Similar results were obtained by flow cytometry (Figure 7, A). Statistical analysis showed that the LC3-II protein expression was significantly higher (p <0.05) in the group with severe asthma (Figure 7, B).

The LC3 protein is present in the cytosol of most cells and exists in three isoforms (A, B and C). When autophagy is triggered, the protein is released and cleaved into LC3-I and LC3-II protein. These proteins could be used as markers in the detection of the different phases of autophagy.

This present aimed to investigate the morphological and biochemical features of autophagy in peripheral blood T-cells (PBTC) from asthmatic patients and healthy donors in stress conditions. In asthma, T lymphocyte persistence may play a key role in the pathophysiology that leads to chronic persistent airway inflammation. It is known from previous study that the apoptosis in PBTC from asthmatic patients was inhibited ^{25, 26}. What about autophagy?

• Involvment of autophagy in Asthma

The results showed that autophagy was activated in T-cells of patients with MA and SA compared to the control patients. Since autophagy contributes to cell survival during the stress condition, fresh isolated T-cells were kept in the complete culture medium for 72 hours. Prolong culture period led to depletion of nutrients in the media inducing stress. It was observed that autophagosome formation in PBTC of patients with asthma occurred within 72 hours of culture. In contrast, in PBTC from healthy donors the deprivation of nutrients initiated apoptosis. The cells in this studied group had a typical morphology of apoptotic cells. Manifestations of autophagy were different depending on severity of asthma. In the group of mild asthma, it is a stimulus to autophagic cell death because apoptosis was disturbed as it was shown earlier ²⁶. Moreover, autophagic cell death is determined by the accumulation of autophagic vacuoles in the degraded fragments of cells. From the results of TEM, we detected the fragments of cells which contain a lot of autophagosomes, indicating activation of autophagic cell death. Interesting results were obtained from the group of severe asthma. The results showed apoptotic and autophagic changes at the same time and in the same cells. Therefore, in our study we could assume that the decrease of nutrients was a signal for initiation of apoptosis and autophagy, and thus autophagy was an initial step for induction of apoptosis. It confirms the work of Kroemer et al. ²⁷ who showed that autophagy often precedes apoptosis as the last attempt for the cells to escape death. Recent studies also provided important data about the initiator role of autophagy in Apoptosis ^{28, 29}.

LC3-II expression study by fluorescence microscopy showed that T-cells of patients with MA and SA had autophagosomes which expressed LC3-II protein. Similar observations have been made on granulocytes by Ban et al. ¹⁸ who noted Higher autophagy levels in sputum granulocytes, peripheral blood cells and peripheral blood eosinophils from patients with severe asthma than from patients with non-severe asthma and healthy controls (P < 0.05). LC3-II is known as a marker of autophagy ³⁰ and quantification of LC3-II protein is used to quantity autophagosome ³⁰. In our study LC3-II isoforms were established only in T-cells of SA donors. In the group with MA the data obtained by immunoblotting assay differ from electron microscopy study. Because we did not get LC3-II isoforms that does not mean it is absent. One of the hypotheses that could be put forward is that LC3-II itself was probably degraded by autophagy because it was associated with autophagosome membrane and it makes the interpretation of LC3 immunoblotting results problematic. The detection method used could also be indexed.

• Dexamethasone, autophagy and asthma

The effect of dexamethasone on autophagy of PBTC in asthmatic patients and healthy donors was evaluated. We assumed that dexamethasone-treatment stimulates apoptosis in T-cells of patients with SA. Indeed, in the group with mild form dexamethasone stimulates apoptosis, and autophagosomes were not detected. Therefore, we did not establish the conversion of LC3-I to LC3-II by immunoblotting assay. The part of cells did not express LC3-II protein in comparison with cells without treatment. So we can conclude that in the group with MA the dexamethasone treatment promotes apoptosis, but inhibits autophagy. In T lymphocytes of patients with SA, we demonstrated that dexamethasone induces autophagy, but not apoptosis. In other study, It is showed that dexamethasone did not affect autophagy levels in peripheral blood eosinophils ¹⁸.

A study performed in 2008, indicated that dexamethasone treatment induce autophagy in Bcl-2-positive T cell lymphoma line WEHI7.2. and inhibit apoptosis ³¹. Bcl-2 family of proteins are anti-apoptotic ³². As shown in earlier study the anti-apoptotic Bcl-2 protein increased in patients with SA compared to those with MA ³³. but Ying et al. ³⁴, on the other hand, reported a decrease in expression of Bcl-2 on lymphocytes isolated from broncho-alveoar lavage fluid from asthmatic patients, compared with healthy adults. Abdulamir et al. attempts to reconcile the controversy and indicated that the percentage of lymphocytes with Bcl-2 expression depends on asthma severity ³⁵. Thus, we could speculate that, in our study we had two types of patients: the people with mild form of asthma whose T lymphocytes were Bcl-2-negative and the patients with SA, who’s T-cells, are Bcl-2-positive and had overexpression by this gene. This suggests that the Bcl-2 family of proteins not only regulates apoptosis, but also controls cell death that depends on autophagy genes. It could be a reason for different effects of dexamethasone treatment in patients with mild and severe asthma in our study. The hypothesis that dexamethasone induces autophagy in T-cells was confirmed in the group with SA in our study. But this hypothesis needs to be investigated in the future.

4. Conclusion

Autophagy could play an important role in the pathogenesis of asthma especially of severe asthma and may be contribute to the survival and the activation of T-lymphocyte in patients with severe asthma. Our next study will be based on a comparative study of autophagy and apoptosis of lymphocytes in mild and severe weanling asthmatics. This study can also be improved by quantifying the number of autophagosomes and autolysosomes with TEM and studying the dependency relationships between autophagy and the type 1 programmed cell death process. We shall also perform functional assays to better understand the effect of autophagy induction on T cell activation.

List of Abbreviations

MA: Mild persistent Asthma

SA: Severe persistent Asthma

NCs: Normal healthy Controls

Dex: Dexamethasone

PBTC: Peripheral Blood T-Cells

Acknowledgements

We thank Prof. Myctaffin (Kazan Medical Institute) and Prof. BABA-MOUSSA (University of Abomey-calavi) for supporting our discussion and providing technical expertise.

Funding

No financial support was provided.

Ethics Approval and Consent to Participate

The work was performed in accordance with the rules of the Ethics Committee in the laboratory of Clinical Immunology and Allergy of RKB and with the regulations of the Ministry of Health of the Russian Federation in compliance with the Helsinki Declaration (1975). Formal consent have been obtained from participants

Consent for Publication

All authors consent for publications

Availability of Data and Material

All data analyzed during this study are included in this published article.

Competing Interests

We have no competing interests. All authors have read and approved the final manuscript.

References