The potency of lncRNA MALAT1/miR-155/CTLA4 axis in altering Th1/Th2 balance of asthma

Objectives: This study examined if the MALAT1/miR-155/CTLA-4 axis was involved in modifying Th1/Th2 balance, a critical indicator for asthma progression. Methods: Altogether 772 asthma patients and 441 healthy controls were recruited, and their blood samples were collected to determine expressional levels of MALAT1, miR-155, CTLA-4, T-bet, GATA3, Th1-type cytokines and Th2-type cytokines. The CD 4+ T cells were administered with pcDNA3.1-MALAT1, si-MALAT1, miR-155 mimic and miR-155 inhibitor to assess their effects on cytokine release. The luciferase reporter gene assay was also adopted to evaluate the sponging relationships between MALAT1 and miR-155, as well as between miR-155 and CTLA-4. Results: Over-expressed MALAT1 and under-expressed miR-155 were more frequently detected among asthma patients who showed traits of reduced forced expiratory failure volume in 1 second (FEV1), FEV1/forced vital capacity (FVC) and FEV1% of predicted ( P <0.05). Moreover, MALAT1 expression was negatively with the Th1/Th2 and T-bet/GATA3 ratios, yet miR-155 expression displayed a positively correlation with the ratios ( P <0.05). Additionally, the IFN-γ, IL-2 and T-bet levels were reduced under the influence of pcDNA3.1-MALAT1 and miR-155 inhibitor, while levels of IL-4, IL-10 and GATA3 were raised under identical settings ( P <0.05). Furthermore, MALAT1 constrained expression of miR-155 within CD 4+ T cells by sponging it, and CTLA-4 could interfere with the effects of MALAT1 and miR-155 on Th1/Th2 balance and T-bet/Gata3 ratio ( P <0.05). Conclusion: MALAT1 sponging miR-155 was involved with regulation of Th1/Th2 balance within CD 4+ T cells, which might aid to develop therapies for amelioration of asthmatic inflammation.


Introduction
Bronchial asthma, a chronic inflammatory disorder, is clinically manifested as recurrent wheezing, chest distress and cough [1,2]. According to the estimation of World Health Organization (WHO), the global prevalence of asthma has reached up to 300 million, and delayed treatments could increase disease mortality [3,4]. Multiple factors induce asthma, including environment, heredity and immunity [5,6]. Of note, one immunity-centric theory proposed that asthmatic progression was accompanied by Th2-oriented differentiation of T lymphocytes, and the latter favored release of Th2-type cytokines over production of Th1-type cytokines [7,8]. The resultant bias of Th1/Th2 ratio was a trigger of dysfunctional cell immunity and humoral response, which finally led to asthma exacerbation [9,10]. From the above, suppressing T cell activation or elevating the Th1/Th2 ratio might be conducive to relieving asthma-relevant inflammation from the source.
LncRNAs, molecules with a length of more than 200 nucleotides, regulate numerous biological processes in humans [11,12]. The crucial role of lncRNAs in modulating asthma development has also been increasingly stressed, given their involvement in regulating the inflammatory mechanism [13,14] and function of airway smooth muscle (ASM) [15][16][17]. For instance, lipopolysaccharide (LPS)-induced inflammation is enhanced after knock-out of lncRNA interleukin 7 receptor (IL7R) [13], and expression of lncRNA H19 was reduced after stimulation of pro-inflammatory cytokines (e.g. IL-1β and TNF-α) [18]. On the other hand, exposure to fetal calf serum (FCS) and dexamethasone was found to engender expressional changes of LINC00882, LINC00883 and lncRNA PVT1 within ASM cells (ASMCs) [15]. Notably, the lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) investigated here was also potentially relevant to asthma etiology, owing to that MALAT1 was able to aggravate inflammatory reactions via negative modification of miR-125b [19]. MiR-125b was markedly under-expressed Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20190397/865395/bsr-2019-0397.pdf by guest on 08 January 2020 within asthma patients when compared with healthy controls, and it was a sensitive reflector of asthma severity [20]. In addition, under-expression of MALAT1 was discovered to ease symptoms of myocardial inflammation [21], and production of TNF-α and IL-6 in endothelial cells was spurred when MALAT1 expression was intentionally elevated [22,23]. These findings all indicated that MALAT1 was associated with activation of inflammation, though whether MALAT1 indeed affected asthma-related inflammation remained unknown.
Additionally, miRNAs are commonly-acknowledged collaborators of lncRNAs in precise regulation of disease pathogenesis [24], and they are also indispensable modulators for certain inflammatory airway diseases (e.g. asthma) [25]. For instance, the expressional range of miR-155, which was sponged by MALAT1 [26], could mirror evolution of airway inflammation and airway hyper-responsiveness that occurred in ovalbumin (OVA)-sensitized asthmatic mice [27][28][29]. The intrinsic mechanism might be due to the fact that miR-155 regulated differentiation of T cells (e.g. Th2, Th17 and Treg cells) [30][31][32][33] and the antigen-presenting process of dendritic cells (DCs) [34][35][36]. More than that, certain genes (e.g. CTLA-4) were capable assistants of miR-155 in changing the activity of Th cells and thereby in inducing asthma onset [37]. Nevertheless, further data was needed to determine whether MALAT1 combined with miR-155 and CTLA-4 could drive the onset of asthma.
To tentatively tap this field, we attempted to figure out if MALAT1 was capable of altering Th1/Th2 balance in the context of asthma, which might offer a novel direction for preventing asthmatic inflammation.

Inclusion of study subjects
From April 2015 to May 2018, we recruited 772 asthma patients from Affiliated Hospital of Jining Medical University, and they all met the diagnostic criteria for acute attack of asthma [38]. The asthma patients all showed no liver or kidney-related dysfunctions, and they were not plagued by respiratory tract infection (RTI) or heart failure within the past 2 months. Simultaneously, 441 healthy volunteers were incorporated into the control group, and none of them carried infectious disorders within the past 2 months. All the participants have signed informed consents, and procedures of this investigation have gained approval from Affiliated Hospital of Jining Medical University and the ethics committee of Affiliated Hospital of Jining Medical University.

Evaluation of Th1/Th2 balance among asthma patients
About 5 ml venous blood was collected from each asthma patient, and the blood samples were centrifuged at 16000×g for 10 min to acquire the supernatants. Then expressions of Th1-type cytokines (i.e. IL-2 and IFN-γ) and Th2-type cytokines (i.e. IL-4 and IL-10) were measured according to the guidance of respective enzyme-linked immuno-sorbent assay (ELISA) kits (ZSBIO, Beijing, China), and the Th1/Th2 ratio was calculated as per the formula of (Concentration IL-2 +Concentration IFN-γ )/(Concentration IL-4 +Concentration IL-10 ).
Moreover, expressions of T-bet and GATA3 were evaluated through performing western blotting as specified below, and expressions of MALAT1 and miR-155 were determined by means of reverse transcription-polymerase chain reaction (RT-PCR) also as depicted below. All these experiments were repeated for ≥ 3 times.

Separation of CD 4+ T cells
Approximately 5 ml peripheral blood was gathered from healthy volunteers, with ethylene diamine Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20190397/865395/bsr-2019-0397.pdf by guest on 08 January 2020 tetraacetic acid (EDTA)-K2 added in advance for anti-coagulation. Then, peripheral blood mononuclear cells (PBMCs) were isolated under the weight of lymphocyte separation medium (Sangon, China). After rinsing the PBMCs with phosphate buffer (PBS) for twice, the CD 4+ T cells were separated through a magnetic activated cell sorting (MACS) system (Miltenyi Biotech, German). The specific procedures were summarized as: 1) every 1×10 8 cells were mixed with 900 μl solution that was composed of 2 mmol/L EDTA, 20 mL/L FBS and PB; 2) 100 μl CD 4 microbeads (Miltenyi Biotech, German) were supplemented proportionally; 3) incubation was sustained in the dark for 20 min, followed by rinse of LS column with 3 ml of solution; 4) 1000-r/min centrifugation was performed for 10 min after addition of 10 ml of solution; 5) the resultant supernatants were removed before re-suspension of cell sediments in 2 ml of solution; 6) the cells that dropped from MACS were CD 4-T cells; 7) LS column was transferred onto the centrifugal tube and was washed with 5 ml of solution; and 8) the cells finally collected were CD 4+ T cells. All these experiments were repeated for ≥ 3 times.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from serum and CD 4+ T cells with aid of Trizol reagent (TIANGEN, China), and its integrity was confirmed by polyacrylamide gel electrophoresis (PAGE

Dual-luciferase reporter gene assay
The MALAT1 and CTLA-4 fragments that contained target sites of miR-155 were amplified through conduction of PCR. The products were then cloned into pRL-TK/Rluc plasmids (Promega, USA) to establish pRL-TK/Rluc-MALAT1 wide type (Wt) and pRL-TK/Rluc-CTLA-4 Wt. The the dual-luciferase ® reporter assay system (Primega, USA). All these experiments were repeated for ≥ 3 times.

Statistical analyses
All the statistical analyses were implemented with usage of SPSS software v17.0 and GraphPad Prism v5. The measurement data, which were manifested as mean±standard deviation (SD), were compared based on student's t test or analysis of variance (ANOVA). The categorical data in the form of n or percentage (%) were analyzed by adopting chi-square test. It was regarded as statistically significant in case that P value was less than 0.05.

Association of MALAT1/miR-155 expression with clinical traits of asthma patients
The asthma patients and healthy controls were well matched in their mean age, sex ratio, body mass index (BMI) and smoking history (P>0.05) and, as expected, a higher proportion of asthma history was observed among asthma patients when compared with healthy controls ( 2 =10.72, P=0.001) ( Table 2). Moreover, production of nitric oxide (FeNO) was greater in asthma subjects as compared with healthy people (P<0.05). The healthy population had a higher forced expiratory volume in 1 second (FEV1), FEV1/forced vital capacity (FVC) and FEV1% of predicted when compared with asthma patients (P<0.05). In addition, differential expressions of MALAT and miR-155 were also observed between asthma patients and healthy controls (P<0.05) ( Figure 1A). With median MALAT1 expression as the dividing line, the recruited asthma patients were grouped into those with highly-expressed MALAT1 and those carrying lowly-expressed MALAT1. In the same manner, the highly-expressed (>median) miR-155 group and lowly-expressed (≤median) miR-155 group were also drawn from the identical asthma group (Table 3). It was interesting to observe that the asthma patients who carried lowly-expressed MALAT1 and highly-expressed miR-155 were more prone to reveal higher FEV1 (>1.95 L), FEV1/FVC (>71.55%), predicted FEV1% (>78.91%) and FeNO (≤51.27 μg/L) than highly-expressed MALAT1 group and lowly-expressed miR-155 group (P<0.05).

CTLA-4 interfered with the impacts of miR-155 and MALAT1 on Th1/Th2 balance and T-bet/Gata3 ratio within CD 4+ T cells
The binding sites between miR-155 and CTLA-4 were speculated based on miRBase software

Discussion
Airway inflammation induces reversible airway obstruction and airway hyper-responsiveness, which are key asthma features. The CD 4+ T cells are major participators in asthma-relevant inflammation [39], and the skewing of T cells into Th2 cells causes an imbalance of Th1-type and Th2-type cytokines, which promotes the onset and progression of asthma [40,41]. Since the transcription factors, such as T-bet and GATA-3, enhance the production of Th1-type cytokines (e.g.

IFN-γ) [42]
and Th2-type cytokines (e.g. IL-4 and IL-5), respectively [43], the T-bet/GATA3 ratio investigated here ( Figure 1E) are viewed as a reflection of Th1/Th2 balance [44]. Based on the findings, the Th1/Th2 balance and T-bet/GATA3 ratio that varied within CD 4+ T cells appeared as reliable indicators of asthma progression, and in this investigation we focused on them as markers of dys-regulated inflammation underlying asthma etiology.
Our results showed that both the Th1/Th2 ratio and the T-bet/GATA3 ratio were markedly reduced within asthma patients when compared with healthy controls ( Figure 1B, Table 2), and there were negative correlations between MALAT1 expression and these ratios ( Figure 1C). We partly ascribed this association to the fact that up-regulated MALAT1 expression could enable production of Th2-type cytokines and simultaneously curb release of Th1-type cytokines by CD 4+ T cells ( Figure 2). Actually, the role of MALAT1 in modulating inflammation, though hidden, has been implicated before. For instance, over-expressed MALAT1 was found to increase expression of p38 MAPK and NF-κB [19], whose activities were intensified in mice models of asthma [45,46].
Beyond that, p38 MAPK and NF-κB both could boost production of asthma-related cytokines, established a link between MALAT1 and Th1/Th2 imbalance-triggered inflammation, which was also validated in asthma cell models here (Figure 2).
Our findings also showed that MALAT1 was capable of negatively modulating miR-155 expression in CD4+ T cells by sponging it ( Figure 3A and 3B Figure 1D). We strived to explain this divergence from following two aspects. In the first place, the asthma mice models adopted by Another noteworthy point of this study lied in that MALAT1 affected expression of Th1-type and Th2-type cytokines more strongly than miR-155 (Figure 2), which could be ascribed to that . Apart from that, the CTLA-4 also presented a negative feedback effect against aggravated inflammation [58,59], which suggested that the failure to sustain high CTLA-4 expression might make asthmatics unable to suppress Th2-cell differentiation. Adding to these prior observations, our study shows for the first time that CTLA-4 regulates asthma inflammation by modifying the effects of MALAT1 and miR-155 ( Figure 3D and 5A).
In conclusion, our study indicates that MALAT1 sponging miR-155 could alter the Th1/Th2 balance within CD 4+ T cells through a CTLA-4-dependent mechanism. Further studies are needed to determine whether targeting the MALAT1/miR-155/CTLA-4 axis could alter the Th2 responses that promote airway inflammation in asthma.