Transfer of Invitro CD4 + T Cells with Hypomethylation of Perforin Promoter into Rats’ Abdomens Causes Autoimmune Emphysema

Abstract

Our previous study suggested that hypomethylation of perforin promoter of CD4 + T cells might be involved in the pathogenesis of autoimmune emphysema of rats. Whether transfer of this kind of cells hypomethylated in vitro into naive immunocompetent rats also results in emphysema is unknown yet. To test the hypothesis above, thirty Sprague Dawley (SD) rats were randomly divided into three groups: a model group (n = 10), a normal control group (n = 10) and a sham operation group (n = 10). In the model group, spleen-derived CD4 + T cells of normal rats were treated with 5-azacytidine (5-Aza), complete Freund’s adjuvant and Phosphate Buffered Saline (PBS), then transferred into naive immunocompetent rats. The normal control group was injected with CD4 + T lymphocytes from spleens of normal rats and the same amount of adjuvant and PBS as above. In sham operation group, normal rats were injected intraperitoneally with complete Freund’s adjuvant and PBS. Histopathological evaluations (mean linear Intercept (MLI) and mean alveolar numbers (MAN)), anti-endothelial cell antibodies (AECA) in serum and bronchoalveolar lavage fluid (BALF), lung vascular endothelial growth factor (VEGF)), the apoptotic index (AI) of alveolar septal cells and the methylation levels of perforin promoter of CD4 + T cells were investigated. The levels of the methylation above and MAN were lower in the model group than in the control and the sham operation group, while the AECA in serum and BALF, VEGF, MLI and the AI were greater (all p < 0.05). The methylation levels of perforin promoter were positively correlated with the MAN (r = 0.747, p < 0.05) and negatively correlated with AI, AECA, MLI, and VEGF (r was −0.789, −0.746, −0.743, −0.660, respectively, all p < 0.05). This study suggests that transfer of invitro CD4 + T cells with hypomethylation of perforin promoter into rats causes autoimmune emphysema, possibly by increasing expression of VEGF and promoting alveolar septal cell apoptosis.

Keywords:

• DNA methylation invitro CD4 + T cells
• perforin promoter
• alveolar septal cell apoptosis
• autoimmune emphysema

Introduction

Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). The exact mechanisms that prompt the development and progression of this disease are not clear yet. Cigarette smoking is a major risk factor for the development of COPD, which results in pulmonary chronic inflammation, oxidative stress, and protease/antiprotease imbalance, thereby causing lung injury and emphysema [1].

But nonsmokers may also develop chronic airflow restriction [2]. It is believed that less than 50% of heavy smokers suffer from COPD [3], and the alveolar destruction associated with airspace enlargement usually progresses in severe COPD patients, although they have quitted smoking for many years [4]. Previous studies have demonstrated that autoimmunity has been suspected to relate to the development of COPD [5] and that cellular immunity induced by T lymphocytes is involved in the pathogenesis of this disease [6, 7], suggesting the possible role of T cells in COPD -emphysema.

Vascular endothelial growth factor(VEGF), an obligatory endothelial cell (EC) survival factor abundantly expressed in lung tissues, serves as a critical lung structure maintenance factor [8] and anti-endothelial cell antibodies (AECA) is a heterogeneous group of autoimmune antibody. Related literatures have showed that the abnormal expression of VEGF and AECA plays pathogenic roles in vascular endothelial cells activation and induction of cell apoptosis [9, 10]. In recent years, previous studies have found that the imbalance of apoptosis and proliferation of alveolar septal cells including alveolar epithelial cells and vascular endothelial cells also results in the damage of lung tissues and the formation of emphysema. Therefore, some scholars even call it the fourth pathogenesis of COPD-emphysema [11, 12]. Since one study showed intraperitoneal injection of human umbilical vein endothelial cells into immunocompetent rats caused AECA humoral response, apoptosis of alveolar septal cells and emphysema and that adoptive transfer of CD4+ T spleen cells isolated from the emphysema rats above into naive immunocompetent rats resulted in emphysema, too [8]. We think CD4 + T cell–dependent mechanisms are sufficient to trigger the development of emphysema. Based on our previous study indicating that hypomethylation of perforin gene promoter in CD4+ T cells and the apoptosis of alveolar septal cells might be involved in the pathogenesis of autoimmune emphysema in rats [13], we speculate that hypomethylation of perforin promoter in CD4+ T cells may induce increase of AECA and apoptosis of alveolar septal cells associated with the development of experimental autoimmune emphysema.

5-azacytidine (5-Aza) is a DNA methyltransferase inhibitor. Treating CD4+ T cell clones, as well as polyclonal CD4+ T cells in vitro, with DNA methylation inhibitors caused changes of its reactivity and function. Since transfer of pathogenic CD4 + T cells caused autoimmune emphysema [8], we hypothesize that transfer of CD4 + T cells hypomethylated may induce autoimmune emphysema of rats.

In this study, we used 5-Aza to treat CD4 + T cells of rats in vitro, and then injected normal naive immunocompetent rats intraperitoneally with them to further investigate the role of hypomethylated CD4 + T cells in experimental autoimmune emphysema. Our study showed transfer of the cells above into rats caused autoimmune emphysema to cast light on the possible immunological pathogenesis of this kind of disease.

Methods

CD4+T cells culture and 5-Aza intervention

Experiments were approved by the Animal Ethics Committee of Guizhou Provincial People’s Hospital (ethics IRB numbers: 2015011). Spleen-derived CD4 + T lymphocytes from normal naive immunocompetent Sprague Dawley (SD) rats were isolated by MACS1 magnetic beads as previously described[8]. The purity of enriched CD4+T cell isolates was evaluated by flow cytometry and generally more than 94%. Then, part of them were incubated in cell culture medium containing 5-Aza for 72 h (5-Aza concentration was 10 μmol/l). Those cells (Cell group 1, n = 10) were used to establish emphysema models. Other spleen-derived CD4 + T lymphocytes, not treated by 5-Aza (Cell group 2, n = 10) were also incubated for 72 h.

DNA extraction and bisulfite amplicon sequencing in cell group 1 and cell group 2

In Cell group 1 and Cell group 2, DNA extraction of rats’ spleens was performed as described previously [14], and then bisulfite next-generation sequencing (NGS) was carried out to investigate the perforin gene promotor methylation [15]. The degree of methylation is expressed as the percentage of total cytosines methylated at individual CpG sites. For most CpG sites, hundreds of reads per sample were used for each percentage methylation determination. In all cases, at least 10 reads/CpG site/samples were used.

Animals and CD4 + T lymphocytes hypomethylated

Fifty male SD rats were purchased from Chongqing Tengxin Technology Co., Ltd. (weight range between 180 and 200 g, 6 weeks old). Thirty rats were randomly divided into a model group (n = 10), a normal control group (n = 10) and a sham operation group (n = 10). The model group was injected intraperitoneally with cells (1 × 10⁷) from cell group 1, complete Freund’s adjuvant (1 ml) and PBS (1 ml). The normal control group was injected intraperitoneally with cells (1 × 10⁷) from cell group 2, and the same amount of adjuvant and PBS as above. The sham operation group was also injected intraperitoneally with the same amount of adjuvant and PBS as above.

Morphological assessment of lung tissues

At day 21 of the experiment, all rats were anesthetized with chloral hydrate (3 ml/kg) intraperitoneally. Then the rats were killed. The right lungs were filled with 4% paraformaldehyde for half an hour before fixed in paraformaldehyde for twenty-four hours and then stained with hematoxylin-eosin (HE). The pathological changes were observed under a light microscope. The mean linear intercept (MLI) and the mean alveolar numbers (MAN) of lung tissues were determined as described previously [13, 16]. MLI, as a measure of interalveolar wall distance, was determined by light microscopy at a total magnification of ×100. It was calculated for each sample based on 15 random fields using a cross-line. The total length of the cross-line divided by the numbers of the alveolar walls intersecting the test lines was defined as MLI. MAN, an indicator of alveolar density, was calculated for each sample based on 15 random fields by counting the numbers of alveoli in each field and dividing this number by the area of the field.

Bronchoalveolar lavage and serum analysis

Bronchoalveolar lavage fluid (BALF) and serum were prepared as described previously [16]. BAL was performed as follows: 3 ml aliquots of normal saline were slowly infused in the lungs through tracheostomy, at a constant pressure of 25 cm H₂O, and then withdrawn gently. The lavage was repeated three times using the same syringe. BALF and serum were collected and centrifuged at 1000 rpm for 5 min. Then, the supernatants were stored at −70 °C. The levels of AECA were determined using enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s protocol.(Abbkine Scientific Co., Ltd, USA).

Immunohistochemistry

Expression of VEGF in rat lung was detected by immunohistochemistry according to the manufacturer’s protocol. (Servicebio, WuHan, China). Image Pro Plus 6.0 (Media Cybernetics Company) was used to determine the average optical density(OD) value.

Quantitative analysis of apoptosis

Alveolar septal cell apoptosis was detected by Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) technique (In Situ Apoptosis Detection Kit; Roche, Basel, Switzerland). The total alveolar septal cells and the number of positive alveolar septal cells were counted by one observer. The AI was calculated as described previously [16]. The percentage of positive alveolar septal cells of the total number of alveolar septal cells was calculated for each image and a mean value was obtained for each rat. The results were expressed as the apoptotic index (AI).

DNA extraction and bisulfite amplicon sequencing in three groups

Spleen-derived CD4 + T lymphocytes from the model group, the normal control group and the sham operation group were isolated by MACS1 magnetic beads as previously described [8]. The purity of enriched CD4+ T cell isolates was evaluated by flow cytometry and generally more than 94%. Then the DNA extraction and bisulfite amplicon sequencing were performed as above.

Statistical analysis

SPSS 22.0 statistical software (IBM, USA) was used for data analysis. The data were expressed as the Mean ± standard deviation. Differences were evaluated for statistical significance using analysis of variance and Student’s t test. Pearson correlation was used. P values < 0.05 were considered to be statistically significant.

Results

Methylation status of perforin gene promoter region in Cell group 1 and Cell group 2

The degree of purity of CD4+ T cell isolated was more than 94% as tested by flow cytometry. The methylation levels of the perforin gene promoters of CD4 + T lymphocytes were shown in Table 1. The methylation levels were lower in Cell group 1 than in the Cell group 2 ((0.8186 ± 0.0482) of Cell group 1 vs (0.9316 ± 0.0234) of Cell group 2, p < 0.05, Table 1).

Table 1. The methylation levels of the perforin gene promoter in CD4 + T cells of the two Cell groups.

Groups	N	Methylation level
Cell group 1	10	0.8186 ± 0.0482^a
Cell group 2	10	0.9316 ± 0.0234
T value		−6.66
P value		<0.05

Notes: DNA extraction of rats’ spleens was performed and NGS was carried out. Methylation levels of the perforin gene promoter. ap < 0.05, as compared with the Cell group 2.

NGS, next-generation sequencing.

Histopathological alteration

The lung tissues were normal in the normal control group (Figure 1A) and the sham operation group (Figure 1B), while the model group had enlarged airspace and a loss of alveolar septa showing noticeable emphysema (Figure 2C). The MLI was significantly greater while the MAN was lower in the model group than in the control group (MLI: (47.79 ± 16.83)×10⁻⁶m of model group vs (34.75 ± 6.90)×10⁻⁶m of control group; MAN: (322.31 ± 90.05)×10⁶/m² of model group vs(467.28 ± 59.97)×10⁶/m² of control group) and sham operation group (MLI:(47.79 ± 16.83)×10⁻⁶m of model group vs (32.69 ± 6.65)×10⁻⁶m of sham operation group; MAN:(322.31 ± 90.05)×10⁶/m² of model group vs (470.36 ± 60.06)× 10⁶/m² of sham operation group, p < 0.05, Table 2).

Figure 1. Histology of lung tissue. Notes: Lung tissue section was stained with HE. (A) Control rats receiving normal CD4 + T cells, adjuvant and PBS; (B) Rats receiving adjuvant and PBS; (C) Rats receiving CD4 + T cells hypomethylated, adjuvant and PBS. Data presented were one representative image data. Magnification: 400×. Abbreviations: HE, hematoxylin-eosin; PBS, phosphate buffered saline.

Figure 2. Expression of VEGF in rat lungs Notes: Expression of VEGF in rat lungs was detected by immunohistochemistry. (A) Control rats receiving normal CD4 + T cells, adjuvant and PBS; (B) Rats receiving adjuvant and PBS; (C) Rats receiving CD4 + T cells hypomethylated, adjuvant and PBS. Data presented were one representative image data. Magnification: 400×. Abbreviations: VEGF, Vascular endothelial growth factor; PBS, phosphate buffered saline.

Table 2. The difference of MLI and MAN in the three groups.

Groups	n	MLI (×10^-6m)	MAN (×106/m^2)
Control group	10	34.75 ± 6.90	467.28 ± 59.97
Sham operation group	10	32.69 ± 6.65	470.36 ± 60.06
Model group	10	47.79 ± 16.83a,b	322.31 ± 90.05a,b
F value		5.359	14.02
P value		<0.05	<0.05

Notes: Lung tissue section was stained with HE. MLI and MAN. ^ap < 0.05, as compared with the control group, ^bp < 0.05, as compared with the sham operation group.

MLI, mean linear intercept; MAN, mean alveolar numbers; HE, hematoxylin-eosin.

Comparison of AECA levels

The levels of AECA were higher both in serum and BALF in the model group than in the control group and the sham operation group(AECA in serum: (60.93 ± 12.58) ng/ml of model group vs (27.27 ± 6.41)ng/ml of control group; (60.93 ± 12.58) ng/ml of model group vs (23.82 ± 6.25) ng/ml of sham operation group; AECA in BALF: (41.57 ± 13.49) ng/ml of model group vs (19.52 ± 9.80) ng/ml of control group; (41.57 ± 13.49) ng/ml of model group vs (16.11 ± 7.83) ng/ml, p < 0.05, Table 3).

Table 3. The difference of AECA in the three groups.

Groups	n	AECA in serum (ng/ml)	AECA in BALF (ng/ml)
Control group	10	27.27 ± 6.41	19.52 ± 9.80
Sham operation group	10	23.82 ± 6.25	16.11 ± 7.83
Model group	10	60.93 ± 12.58a,b	41.57 ± 13.49a,b
F value		52.89	16.89
P value		<0.05	<0.05

Notes: Serum and BALF were collected and ELISA was performed. AECA. ^ap < 0.05, as compared with the control group, ^bp < 0.05, as compared with the sham operation group.

BALF, Bronchoalveolar lavage fluid; ELISA, enzyme linked immunosorbent assay; AECA, anti-endothelial cell antibodies.

Comparison of VEGF levels

The mean optical density of VEGF in lung tissues was significantly greater in the model group than in the control group and the sham operation group (VEGF: (20.43 ± 5.90) of model group vs (14.09 ± 2.67) of control group; (20.43 ± 5.90) of model group vs (13.99 ± 3.47) of sham operation group, p < 0.05, Table 4, Figure 2A–C), indicating a high level of expression of VEGF.

Table 4. The difference of VEGF OD in lung tissues in the three groups.

Groups	n	VEGF OD
Control group	10	14.09 ± 2.67
Sham operation group	10	13.99 ± 3.47
Model group	10	20.43 ± 5.90a,b
F value		7.56
P value		<0.05

Notes: Lung tissue was sectioned and immunohistochemistry was carried out. VEGF.

^ap < 0.05, as compared with the control group, ^bp < 0.05, as compared with the sham operation group.

VEGF, Vascular endothelial growth factor; OD, optical density.

Quantitative analysis of cell apoptosis in alveolar septa

The apoptosis of alveolar septal cells was detected by TUNEL in the lung tissues of the three groups. In the model group, there were more stained cells, namely apoptotic cells in the alveolar septa (Figure 3A) while fewer cells were stained in the control group and the sham operation group (Figure 3B and C). The AI was higher in the model group than in the control group and the sham operation group. ((21.91 ± 8.83)% of model group vs (8.62 ± 3.56)% of control group; (21.91 ± 8.83)% of model group vs (7.82 ± 2.80)% of sham operation group, p < 0.05, Table 5).

Figure 3. Apoptosis of alveolar septal cells in rat lungs. Notes: Apoptosis of alveolar septal cells in rat lungs was detected by immunohistochemistry. (A) Rats receiving CD4 + T cells hypomethylated, adjuvant and PBS; (B) Control rats receiving normal CD4 + T cells, adjuvant and PBS; (C) Rats receiving adjuvant and PBS. Data presented were one representative image data. Magnification: 400×. PBS, phosphate buffered saline.

Table 5. The difference of AI of alveolar septal cells in the three groups.

Groups	n	AI (%)
Control group	10	8.62 ± 3.56
Sham operation group	10	7.82 ± 2.80
Model group	10	21.91 ± 8.83a,b
F value		19.08
P value		<0.05

Notes: Lung tissue was sectioned and TUNEL was carried out. AI. ^ap < 0.05 as compared with the control group, ^bp < 0.05, as compared with the sham operation group.

AI, apoptotic index; TUNEL, Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling.

Methylation status of perforin gene promoter region in the three groups

The degree of purity of CD4+ T cell isolated was more than 94% as tested by flow cytometry. The methylation levels of the perforin gene promoters of CD4 + T lymphocytes were shown in Table 6. The methylation levels of the model group were lower than those of the normal group and the sham operation group ((0.8610 ± 0.0243) of model group vs (0.9346 ± 0.0192) of control group; (0.8610 ± 0.0243) of model group vs (0.9364 ± 0.0217) of sham operation group, p < 0.05, Table 6).

Table 6. The methylation levels of the perforin gene promoter in CD4 + T cells in the three groups.

Groups	N	methylation levels
Control group	10	0.9346 ± 0.0192
Sham operation group	10	0.9364 ± 0.0217
Model group	10	0.8610 ± 0.0243a,b
F value		38.77
P value		<0.05

Notes: DNA extraction of rats’ spleens was performed and NGS was carried out. Methylation levels of the perforin gene promoter. ^ap < 0.05, as compared with the control group, ^bp < 0.05, as compared with the sham operation group.

NGS, next-generation sequencing.

Correlation analysis

In the model group, the methylation levels of perforin gene promoter in CD4+ T cells were positively correlated with the MAN(r = 0.747, p < 0.05) but negatively correlated with the AI(r = −0.789, p < 0.05), the AECA levels in serum and BALF (r = −0.746, −0.661, respectively, p < 0.05)), the MLI (r = −0.743, p < 0.05) and the VEGF OD (r = −0.66, p < 0.05).

Discussion

As we know, the inflammation in COPD patients remains to be persistent and progressive even if they have stopped smoking. Some related literatures suggested that autoimmune response might be involved in the development of COPD-emphysema [17], but the details are not clear yet and further study is needed.

Taraseviciene-Stewart et al. [8] established an autoimmune emphysema rat model by intraperitoneal injection of xenogeneic endothelial cells in the early twenty first century for the first time. They also found adoptive transfer of pathogenic spleen-derived CD4+ T cells into naive immunocompetent rats resulted in emphysema with increased concentration of AECA in vivo, which suggests CD4 + T cell–dependent mechanisms are sufficient to trigger the development of autoimmune emphysema in rats.

DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. It plays an important role in normal development in many higher organisms, usually occurring in the CpG islands, a CG-rich region, upstream of the promoter region. As a powerful methylation inhibitor, 5-Aza can inhibit the growth and proliferation of tumor cells, induce cells’ differentiation, and change the responsiveness and function of T cells [18, 19]. Related studies also showed that treated with 5-Aza in vitro, CD4 + T lymphocytes were hypomethylated and could be made autoreactive [20]. Since transfer of pathogenic CD4+ T cells into naive immunocompetent rats caused emphysema [8], we speculate that CD4+ T cells with hypomethylation of perforin promoter may result in autoimmune mechanisms associated with autoimmune diseases.

Our previous study showed that the promoter of perforin gene in spleen-derived CD4 + T lymphocytes was hypomethylated in autoimmune emphysema in rats [13], indicating that the gene hypomethylation was involved in the pathogenesis of this disease in rats. So, we hypothesize that transfer of this kind of cells hypomethylated in vitro into normal rats may cause emphysema too.

In order to test the hypothesis above, we first treated spleen-derived CD4 + T cells with 5-Aza in vitro and showed that the methylation levels of perforin promoter in spleen-derived CD4 + T cells were lower in the cell group 1 than in the cell group 2, which confirmed the effect of 5-Aza on methylation of perforin gene promoter in CD4 + T cells. Then, we injected normal rats intraperitoneally with the hypomethylated CD4+ T cells and found that the AECA and the MLI were greater in the model group than in the normal group and the sham operation group, while the MAN and the perforin promoter methylation in CD4 + T cells became lower, which showed transfer of those 5-Aza-treated cells was sufficient to induce autoimmune response in rats. Our results further suggested the hypomethylation of perforin promoter in CD4 + T cells was one of important mechanisms to trigger autoimmune emphysema in rats, thus providing new insights into this disease.

Apoptosis, a type of cell death mechanism, is controlled by the interactions between several molecules and responsible for the elimination of unwanted cells from the body [21]. It is wildly accepted that the imbalance of apoptosis and proliferation of alveolar septal cells is implicated in the pathogenesis of emphysema [22], which has captured more attention from all over the world.

VEGF is abundantly expressed in the normal lung, which plays a critical role in the development, growth, and survival of blood vessels. Some of previous literatures have showed that the expression of VEGF is decreased, which induces the apoptosis of alveolar septal cells [23, 24] associated with COPD-emphysema, but related studies demonstrated that it was also involved in the process of small airway remodeling in emphysema rats and emphysema patients [24, 25]. As a result, some scholars even speculated that VEGF might play different roles in different stages of emphysema [26]. At the early stage, VEGF increases to promote the formation of airway epithelia and vascular endothelial cells, while at the late stage, it decreases due to the serious destruction of lungs.

This study showed the expression of VEGF in lung tissues and AI of alveolar septal cells were significantly increased in the model group as compared with the normal group and that the methylation levels of perforin promoter were positively correlated with the MAN and negatively correlated with AI, AECA, MLI and VEGF . So, we speculate that there is a compensatory increase of VEGF to repair the injury at the early stage of the disease induced by adoptive transfer of hypomethylated CD4 + T cells and that increased apoptosis of alveolar septal cells may also contribute to the pathogenesis of autoimmune emphysema in rats.

However, further clinical trials are needed to investigate the role of hypomethylation of perforin promoter in CD4 + T cells in emphysema and the effect of methyl donors on this disease to provide a potential new therapeutic option for the prevention of this disease in humans.

Conclusion

Transfer of invitro CD4 + T cells with hypomethylation of perforin promoter into rats’ abdomens causes autoimmune emphysema, possibly by increasing expression of VEGF and promoting alveolar septal cell apoptosis.

Abbreviations:
SD	=	Sprague Dawley
5-Aza	=	5-azacytidine
PBS	=	Phosphate buffered saline
MLI	=	Mean linear Intercept
MAN	=	Mean alveolar numbers
AECA	=	Antiendothelial cell antibodies
BALF	=	Bronchoalveolar lavage fluid
VEGF	=	Vascular endothelial growth factor
AI	=	Apoptotic index
COPD	=	Chronic obstructive pulmonary disease
EC	=	Endothelial cell
HE	=	Hematoxylin-eosin
TUNEL	=	Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by grants from Guizhou Provincial Science and Technology Foundation (QIAN KE HE basis, ZK[2021], General, No. 350), National Natural Science Foundation of China (No. 81560012, 8176010286, 81860008), the Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences 2019PT320003, Science and Technology Fund of Guizhou Provincial Health Commission (gzwjkj2020-1-049).