Attenuated lncRNA NKILA Enhances the Secretory Function of Airway Epithelial Cells Stimulated by Mycoplasma pneumoniae via NF-κB

The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.


Introduction
Mycoplasma pneumoniae (MP) is a major cause of community-acquired pneumonia, especially in children and young adults [1]. Although MP infection is typically self-limiting, more and more cases have been reported to progress to severe, refractory, and even life-threatening pneumonia [2]. Accumulating evidence demonstrated that excessive airway inflammation plays an essential role in the development of Mycoplasma pneumoniae pneumonia (MPP) [3][4][5].
Airway epithelial cells are barrier cells on the surface of the airways, which have active secretory functions. When activated by external stimuli such as infection, they release a variety of cytokines, thus playing a key role in airway inflammation [6]. Excessive production of cytokines can aggravate pulmonary injury through worsening damage to the respiratory epithelium cells characterized by loss of cilia, vacuolation, and exfoliation [7]. Cytokine secretion is a complex process, in which nuclear factor-κB (NF-κB), an important inflammatory signaling pathway component, plays pivotal roles [8]. According to previous research, the lipoprotein components of MP can recognize toll-like receptors and then activate NF-κB to regulate the secretion of various inflammatory mediators and chemokines, thus generating airway inflammation [9]. Among multiple inflammatory cytokines, interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) have been reported to be prominent for the clinical evaluation of MPP [10].
Long noncoding RNAs (lncRNAs) are a subtype of noncoding transcripts with more than 200 bases of length, which can regulate a variety of physiological and pathological processes [11,12]. lncRNAs may act as enhancers or inhibitors of inflammatory transcription to regulate the dynamics and epigenetics of this process. NF-κB interacting lncRNA (NKILA), which is associated with the NF-κB signaling pathway, has been reported to inhibit multiple inflammatory diseases [13][14][15]. However, whether NKILA could act as a regulator of inflammation in MPP has not been reported to date.
In this study, we aimed to investigate the potential role of NKILA in airway inflammation of children with MPP and the effect of NKILA on the secretory function of airway epithelial cells in vitro.

Study Subjects.
Children with MPP admitted to the Children's Hospital of Nanjing Medical University from September 2018 to December 2018 were enrolled in this study. Children with intrabronchial foreign bodies (FB) were included as controls. The diagnosis of FB depends on the history of airway foreign body inhalation and bronchoscopy results. The diagnosis of pneumonia is based on clinical manifestations including fever, cough, dyspnea, abnormal breathing sounds, and pulmonary imaging abnormalities. MP infection was confirmed by polymerase chain reaction (PCR) for MP in nasopharyngeal secretions and/or serologic testing. Subjects who suffered from chronic underlying conditions, heart diseases, and immune deficiencies or used immunosuppressive drugs were excluded. Besides, the subject's nasopharyngeal secretions needed to test negative for respiratory syncytial viruses, influenza viruses, parainfluenza virus, metapneumovirus, adenovirus, Chlamydia trachomatis, and bacterial cultures. The clinical data are shown in Table 1.

Bronchoscopy and Bronchoalveolar Lavage.
Guidelines for bronchoscopy and alveolar lavage were reviewed in previous literatures [16,17]. We followed the methods described in our recent publication [18]. Children with MPP were subjected to BALF collection within 1 week after admission. For children with FB, the foreign body was removed by bronchoscopy immediately after admission, and BALF specimens were collected during the reexamination of bronchial FB. Lavage was performed with sterile saline solution. BALF was then centrifuged at 1000× g at 4°C for 5 min within 1 h of collection; the supernatant was stored at -20°C, while the precipitate was resuspended in Trizol reagent (Invitrogen, Carlsbad, USA) and stored at -80°C.  M. (Children's Hospital, Zhejiang province, China). The strain was cultured in a Mycoplasma broth, which consists of Mycoplasma broth base (Oxoid, Basingstoke, UK), 0.5% glucose, 0.002% phenol red, and Mycoplasma selective supplement G (Oxoid) as we previously described [18,19]. MP was quantified by counting the number of colony-forming units (CFU) in Mycoplasma agar plates [20]. For in vitro experiments, MP were harvested by centrifugation (10000× g for 20 min), washed, and resuspended in phosphatebuffered saline (PBS) to yield 1 × 10 8 CFU/mL. Cells were incubated with MP solution (100 CFU/mL) for 18 h [21].

Small
Interfering RNA (siRNA) and Cell Transfection. SiRNAs against NKILA and control-scrambled siRNA were designed and synthesized by Ribo Biotechnology Co., Ltd. (Guangzhou, China). Transfection of vectors was performed using Ribo FECT™ CP (Ribo Biotechnology Co., Ltd.) in accordance with the manufacturer's protocol. Sequences of custom siRNA are listed in Table 2. 2.6. Quantitative Real-Time PCR (qRT-PCR) Assay. Total RNA was extracted from cells using Trizol reagent according to manufacturers' instructions (Ambion, Austin, USA). Then RNA was reverse translated to complementary DNA   Primers NKILA: NF-κB interacting lncRNA; F: forward; R: reverse.  (Figure 1(a)). To explore the regulatory role of NF-κB on the secretion of airway inflammatory cytokines in MPP, we detected the concentrations of IL-8 and TNF-α in the BALF of both groups. As shown in Figures 1(b) and 1(c), the levels of IL-8 and TNF-α in BALF were significantly higher in the MPP group compared to the control group. These results demonstrated that NKILA was downregulated in the BALF of

NKILA Knockdown Promoted IκBα Phosphorylation and p65 Translocation in Epithelial Cells.
To study the relationship between NKILA downregulation and NF-κB activation in MP-treated A549 and BEAS-2B cells, siNKILA-1 was utilized to knockdown NKILA and MP treatment was performed. We observed by western blot that the total amount of p65 in total protein extracts of A549 and BEAS-2B cells after NKILA silencing did not change (Figures 3(a) and 3(b)); however, the level of phosphorylated p65 in the cell nucleus extracts was significantly increased (Figures 3(c)  and 3(d)). In addition, the level of phosphorylated IκBα was increased in the cytoplasm of NKILA-depleted epithelial cells after MP infection, while the expression of IκBα was decreased (Figures 3(e) and 3(f)). Taken together, these results demonstrated that NKILA knockdown promoted IκBα phosphorylation and degradation and p65 translocation in MP-treated airway epithelial cells.

NKILA Inhibits NF-κB Activity by Physically Binding to
IκBα. The effects of NKILA on NF-κB activity were examined by the dual-luciferase reporter assay. The results showed that MP treatment increased NF-κB transcriptional activity, while knockdown of NKILA further increased NF-κB activity (Figures 4(a) and 4(b)). To prove that NKILA could physically bind to NF-κB and thereby inhibit its activation, we performed RIP for cell extracts of MP-stimulated A549 cells using the p65 and IκBα antibodies. We observed roughly sevenfold enrichment of IκBα in the immunoprecipitates compared to the IgG control (Figure 4(c)), indicating that NKILA physically associates with IκBα. Based on these results, we believe that NKILA binds to IκBα, thus preventing phosphorylation of IκBα and NF-κB activation in MPinfected airway epithelial cells.

Discussion
IL-8 and TNF-α, known to be two important proinflammatory cytokines, play key roles in inflammation and chemotaxis in the airways caused by MP. Airway epithelium is the main source of IL-8, and lung pathogenesis is known to be correlated with increased IL-8 levels [23]. Besides, elevated TNF-α causes an excessive inflammatory response and induces the release of other inflammatory factors, leading to the damage of cells, tissues, and even multiple organs and systems. In the present study, we focused on the role of NKILA in regulating airway epithelial secretory functions in MPP. Our results showed that NKILA expression was downregulated in BALF of children with MPP; however, the levels of IL-8 and TNF-α were significantly increased in children with MPP compared to children with FB. Therefore, it was speculated that NKILA plays a potential role in regulating airway inflammation in MPP. By conducting in vitro experiments, we showed that MP stimulation upregulated IL-8 and TNF-α in epithelial A549   (e, f) Cytoplasmic IκBα and IκBα phosphorylation levels in NKILA-depleted and control A549 and BEAS-2B cells with or without MP infection were measured by western blot and normalized to β-actin. The samples were derived from the same experiment and gels/blots were processed in parallel. Data are shown as the mean ± SD from three independent experiments ( * * * P < 0:001, * * P < 0:01, and * P < 0:05).

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BioMed Research International and BEAS-2B cells. In order to explore the regulatory role of NKILA in the secretion of cytokines, we knocked down NKILA in epithelial cells and found that silencing of NKILA further increased the levels of proinflammatory cytokines secreted by A549 and BEAS-2B cells treated with MP. Therefore, we can conclude that NKILA plays a negative regulatory role in the inflammation produced by epithelial cells and caused by MP. NF-κB, an important transcription factor involved in the regulation of various inflammatory response genes, is known to play an important role in airway inflammation caused by MP infection. Under physiologic conditions, NF-κB binds to an inhibitor protein named IκB and is retained in the cytoplasm. Upon stimulation, IκB is phosphorylated and degraded and then separated from NF-κB. Further, the phosphorylated NF-κB p65 translocates into the nucleus and activates the transcriptional expression of downstream genes associated with inflammation [24]. According to previous studies, NKILA can block IκB degradation by masking the phosphorylation motifs of IκB and hinder NF-κB translocation [13,15,25,26], thus inhibiting the transcription of downstream inflammatory response genes [27].
In the current study, we observed that MP stimulation induced NF-κB activation in A549 and BEAS-2B cells. Knocking down NKILA facilitated IκBα phosphorylation and degradation and p65 translocation, thus triggering NF-κB activation. Our findings on the regulatory effect of NKILA on NF-κB are consistent with the previous studies [28][29][30]. Additionally, in MP-stimulated A549 cells, we confirmed that NKILA physically associated with IκBα. Liu et al. reported that NKILA physically associates with NF-κB/IκB and blocks IκB phosphorylation, thus inhibiting NF-κB activation in breast cancer cells [13]. However, we did not detect its binding to NF-κB p65. The use of different cell lines and treatment methods could explain these inconsistent results. However, our results suggested that NKILA could negatively regulate NF-κB activation by directly binding to IκBα in MP-treated epithelial cells.
To sum up, we demonstrated that NKILA was downregulated in BALF of children with MPP, while the levels of IL-8 and TNF-α increased. Knockdown of NKILA promoted epithelial cells to secrete MP-induced cytokines. The effects of NKILA on the epithelium were achieved by negative regulation of NF-κB. Therefore, we conclude that attenuating lncRNA NKILA enhances the secretory function of airway epithelial cells stimulated by MP via regulation of NF-κB signaling. Thus, NKILA may serve as a critical therapeutic target for regulating the excessive airway epithelial inflammation caused by MP infection. Our study provides a new theoretical basis for further revealing the inflammatory mechanism of MPP and a potential intervention target for blocking the progression of MPP, which might bring a new perspective to improve the prognosis of children with severe MPP.

Conclusion
Our study revealed that NKILA can affect the secretion function of epithelial cells induced by MP infection via regulation of NF-κB signaling. This finding provides new insights into the molecular mechanisms of inflammation in MPP. NKILA may be a potential therapeutic target for MPP.

Data Availability
The data that support the findings of this study are available from the corresponding author upon request.  Figure 4: NKILA inhibits NF-κB activity by binding to IκBα. (a, b) NF-κB activity of A549 and BEAS-2B cells was detected using a dualluciferase reporter assay. (c) Interaction between NKILA and p65/IκBα revealed by RIP experiments. Cell extracts of MP-stimulated A549 cells were immunoprecipitated with control IgG or anti-p65/IκBα antibody, and the complexes were analyzed for the presence of β-actin mRNA and NKILA by qPCR. Data are shown as the mean ± SD from three independent experiments ( * * * P < 0:001, * * P < 0:01, and * P < 0:05).